An introduction to hydrogen bond scalar couplings · not all, interactions involving biological macromolecules. For both proteins and nucleic acids, ... order of 100!600 kJ mol"1

An Introduction toHydrogen Bond ScalarCouplingsANDREW J. DINGLEY,1,2 FLORENCE CORDIER,3 STEPHAN GRZESIEK3

1 Institute of Physical Biology, Heinrich-Heine-Universitat, 40225 Dusseldorf, Germany¨ ¨2 Institute of Structural Biology, IBI-2, Forschungszentrum Julich, 52425 Julich, Germany¨ ¨3 Department of Structural Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland

( )ABSTRACT: The hydrogen bond H-bond has been recognized in science for more than80 years as a concept to explain situations where a hydrogen atom is simultaneouslybinding to two other atoms. Due to the moderate energies necessary for their formation andrupture, hydrogen bonds play a fundamental role in many chemical reactions and most, ifnot all, interactions involving biological macromolecules. For both proteins and nucleicacids, H-bonds are the essential element in the formation of secondary structures and oftenthey also participate in the stabilization of tertiary structures. Many properties of H-bondshave been studied by a large variety of experimental methods, including NMR spectroscopy.Recently, electron-mediated scalar couplings have been observed which connect magneticnuclei on both sides of the hydrogen bridge. In contrast to earlier NMR observables, thesecouplings can be used to ‘‘see’’ all partners of the hydrogen bond, the donor, the proton,and the acceptor in a single COSY experiment. In addition, the size of the coupling constantcan be related to hydrogen bond distances and angles. This article should serve as anintroduction to these findings and illustrate their use by various examples. ! 2001 JohnWiley & Sons, Inc. Concepts Magn Reson 13: 103!127, 2001

KEY WORDS: DNA; hydrogen bond; nucleic acid; protein; RNA; scalar coupling

INTRODUCTION

Since the first definitive recognition of hydrogen!bonding at the beginning of the last century 1,

Received 6 July 2000; revised 26 October 2000;accepted 26 October 2000.

Correspondence to: Stephan Grzesiek; E-mail: [email protected].

Contract grant sponsor: DFG.Contract grant number: GR1683!1-1.Contract grant sponsor: SNF.Contract grant number: 31-61"757.00.

! . ! .Concepts in Magnetic Resonance, Vol. 13 2 103!127 2001! 2001 John Wiley & Sons, Inc.

. ! .2 , the literature on hydrogen bonds H-bondshas become simply too large to be cited in anyadequate way. Jeffrey and Saenger estimated inthe early 1990s that a paper on H-bonds would be

! .published every half hour 3 . Besides a number! . ! .of monographs 3!11 and review articles 12!18 ,

the chapter on H-bonds in Linus Pauling’s book! .The Nature of the Chemical Bond 19 may serve

as an excellent introduction to this very largescientific field.

A simple succinct definition to hydrogen bond-ing is the weak attraction between a hydrogenatom attached to an electronegative donor atomD and an electronegative acceptor atom A. This

103

DINGLEY, CORDIER, AND GRZESIEK104

force is largely attributed to the electrostatic at-traction between a partial positive charge locatedat the position of the hydrogen atom in theD H group and a partial negative charge onthe acceptor atom A. The predominant types of

. . .H-bonds in biomacromolecules are O H O ,

. . . . . . . . .O H N , N H O , and N H N , al-though weaker H-bonds involving C H groups

! .as donors are also recognized 20!27 . For theseH-bonds, the site of attraction is commonly iden-tified as an electronic lone pair on the oxygen ornitrogen acceptor atom. However, the # elec-trons of aromatic systems can also act as accep-tors, and H-bonds involving sulfur groups ormetallic cofactors are also known.

The overwhelming importance of H-bonds inbiology and chemistry stems from the very moder-ate energies needed for their formation and rup-

! .ture 3 . This makes it possible that H-bonds play!an essential role in many common enzymatic 3,

.9, 28, 29 and chemical reactions occurring inmany common solvents, such as water, at ambienttemperatures. The formation of the H-bonded

! .secondary structures of proteins 30 and nucleic! .acids 31 is probably the most important example

for such a reaction.In contrast to covalent bonds which are of the

order of 100!600 kJ mol"1 in bond energy, theenergies of the usual ‘‘weak’’ H-bonds are ap-

!proximately an order of magnitude smaller i.e.,"1 .# 30 kJ mol . Only the so-called ‘‘very strong’’

H-bonds have bond energies which are similar insize to their covalent counterparts. An example ofsuch a very strong H-bond is the bifluoride ion! ". "1 ! .HF with an energy of 163 ! 4 kJ mol 32 .2In proteins and nucleic acids, the bond energiesof individual H-bonds are often determinedthrough deletion or site-directed mutagenesis! .33!39 , with estimated values ranging between 2and 20 kJ mol"1. However, as the substitution ordeletion of entire residues will also affect otherproperties of the biomacromolecule, the valuesreported from such studies are usually consideredas upper limits. Clear-cut correlations betweenH-bond energy and the H-bond geometry arecurrently missing. On the contrary, it is a veryimportant property of the weak hydrogen bondthat the possible bond distances and bond angles

! .cover a rather wide range 3, 40 . Apparently, theenergy minimum is relatively shallow with respectto a variation of bond distances and angles.Therefore a variety of different H-bond geome-tries can be realized with relative ease. This prop-erty enables the H-bonds to act as a very adaptive‘‘glue’’ and contrasts with covalent bonds that

display distinct preferences with respect to bond! .distances and angles 3 .

In the geometric characterization of a hydro-gen bond, main parameters are the hydrogen

. . . . . .bond distances H A and D A , as well asseveral bond and torsion angles such as the hy-

. . .drogen bond angle D H A . Generally, anH-bond is assumed to be present when the dis-tance R is less than or equal to the sum ofD $ $ $Athe van der Waals radii of the two electronegativeatoms. For example, in the structure of ice, the

˚ ! .R distance of 2.75 A 41, 42 is much smallerO $ $ $O˚than the sum of the van der Waals radii of 3.04 A

! .of the two oxygen atoms 43 . The presence ofH-bonds in biomacromolecular structures is usu-ally inferred from the spatial arrangement of thedonor and acceptor groups once the structure ofa biomacromolecule has been solved by eitherX-ray crystallography, NMR spectroscopy, or evenneutron diffraction. Due to the weak scatteringdensity of the hydrogen atom, it is particularlydifficult to obtain precise information from X-raydiffraction on its position within the H-bond. Only

!for the highest resolution X-ray structures i.e.,˚ ."# 1 A resolution is it possible to ascribe indi-

vidual spatial positions to the hydrogen atomswhich are independent of the use of standardcovalent geometries. Recent technical innova-

! .tions for neutron diffractometry 44!46 bearsome promise to provide more precise informa-tion on the position of either hydrogen or deu-terium nuclei within the H-bonds of biomacro-

! .molecules 47!50 .High resolution NMR has contributed signifi-

cantly to the understanding of H-bonds. Forexample, a large number of different NMR ob-

! .servables 16 , such as chemical shifts, provideindirect evidence for hydrogen bonds. However,in recent years direct evidence has been found bythe presence of scalar couplings between magnet-ically active nuclei on both sides of the hydrogenbridge. Such couplings have been observed both

! .in biomacromolecules 51!76 and in smaller! .chemical compounds 77!80 . The existence of

such scalar couplings makes it possible to corre-late the frequencies of nuclei on both sides of thehydrogen bridge by high resolution NMR experi-ments. Thus, in favorable cases, complete H-bondnetworks in proteins and nucleic acids can beestablished by COSY-type experiments. Besidesthis practical aspect, the size of the couplings hasbeen shown to be influenced by the geometricarrangement of the nuclei involved in the H-bonds. This opens the possibility to derive geo-

AN INTRODUCTION TO HYDROGEN BOND SCALAR COUPLINGS 105

metrical information about the H-bond from theanalysis of the coupling size.

Initially, it was surprising to observe such cou-plings across hydrogen bonds since we usuallyassociate scalar couplings with the presence ofpurely covalent bonds. However, it is now wellestablished that the H-bond scalar couplings fol-low the same electron-mediated polarization

!mechanism as their covalent counterparts 53, 59,.78, 81, 82 . Therefore the same NMR experimen-

tal concepts apply for their detection and quan-! .tification. This introductory review aims to 1

briefly summarize the various NMR observableswhich provide indirect information for individual

! .H-bonds; 2 present a basic theoretical descrip-tion of scalar couplings including the H-bond

! .couplings; 3 illustrate how we originally de-tected the cross hydrogen bond coupling in nu-cleic acids by a systematic investigation of magne-

! .tization losses; 4 discuss the basic experimentalprocedures for measuring various H-bond cou-plings in biomacromolecules. The article con-cludes with a summary of possible applicationsfor the H-bond coupling effect.

INDIRECT NMR OBSERVATIONS OF THEHYDROGEN BOND

Reduced Hydrogen Exchange Rates

Many polar hydrogen atoms, in particular theamide, imino, and hydroxyl group hydrogens inbiomacromolecules continuously exchange withsolvent hydrogens. This continous exchange isslowed down significantly when the hydrogen par-ticipates in an H-bond, even when this bond is at

! .the surface of a biomacromolecule 83!86 .Therefore, hydrogen exchange from an H-bond isgenerally assumed to require H-bond opening.The hydrogen exchange is then modelled as atwo-step mechanism where the first step is theequilibrium reaction between the closed and openH-bond, whereas the second step is the exchange

!reaction from the open state to the solvent 84,.85 . The latter step usually requires the catalysis

by acids or bases. Thus, the hydrogen exchangerates depend on a number of factors such as pH,the pK of the donor group and of the base or acid

! .catalysist s , the catalyst concentration, tempera-ture, solvent accessibility, as well as the H-bond

! .opening rate itself 84, 85, 87 .Proton NMR spectroscopy has quantified many

of the underlying reactions at specific H-bonding! .sites both in nucleic acids 87!89 and polypep-

! .tides 90!92 . Due to the rather low pK of theimino group, around 9, the exchange rates ofH-bonded imino-protons in nucleic acids can beincreased by buffer catalysts with similar pK val-

! .ues, such as ammonia 87 . This increase in ex-change can be used to extrapolate to the situationwhere the base pair opening is the limiting step inhydrogen exchange. Intrinsic opening times forimino H-bonds in Watson!Crick base pairs deter-mined by this method are on the order of mil-

! .liseconds 87!89 . In contrast, catalysis by buffersis ineffective for amide protons in proteins due to

! .the high pK ca. 18.5 of the backbone amide! .group 86 . Therefore, at moderate pH values,

the hydrogen exchange rates in proteins are usu-ally limited by the slow exchange from the openstate of the H-bond to the solvent, and intrinsicopening rates of protein backbone H-bonds can-

! .not be derived 87, 93 . Exchange times for amidehydrogens in short peptides or at the surface ofproteins are on the timescale of milliseconds to

! .seconds 91, 92 . In contrast, H-bonded amidehydrogens located in secondary structure ele-ments have been reported to have exchange times

6 ! .ranging between 10 to 10 min 90 . Clearly, thevery slow exchange rates of amide hydrogens ob-served in the interior of proteins may not only bedue to H-bonding, but also may be caused by

! .limited solvent accessibility 90 .

Chemical Shift

H-bond formation usually results in chemical shiftchanges for all the nuclei involved in the H-bond! .16 . As the chemical shift is intrinsically relatedto the local electronic environment, these chemi-cal shift perturbations indicate a redistribution ofthe electron density upon H-bond formation. ForH-bonding to an electronegative acceptor atomsuch as oxygen or nitrogen, there is always achange in the isotropic chemical shift of the H-bonded hydrogen nucleus to higher frequencies! .downfield shift . This downfield shift is a result ofa number of not yet fully understood and partiallycompeting factors, including a decrease in theelectron density around the hydrogen nucleus anddeshielding effects from the electronic currents ofthe acceptor atom. A large number of examplescan be given for such proton downfield shifts onH-bond formation with oxygen or nitrogen accep-tors; e.g., downfield shifts of the amide proton inproteins have long been recognized to be corre-

! .lated to shorter H-bond lengths 94, 98 . Like-


wise, the formation of H-bonds in nucleic acidbase pairs results in downfield shifts for the imino

! .and amino protons 99, 100 . In certain systems,proton downfield shifts upon H-bond formationhave been observed to be as large as 15!20 ppm! .14 . For example, the extreme chemical shift! .$ 16 ppm of histidine imidazole protons is usedas an indicator for the presence of ‘‘low-barrier’’

! .hydrogen bonds in catalytic processes 101 .Conversely, H-bonding to # electrons in aro-

matic rings usually results in a change of theisotropic 1H chemical shift to lower frequency! .upfield shift . This upfield shift is caused by thearomatic ring current effects outweighing the de-shielding effects from the formation of the H-bond

! .itself 16 .General trends are also observed for the chem-

ical shifts of donor and acceptor nuclei. Invari-ably, in the formation of an H-bond involving anelectronegative acceptor, the donor nucleus expe-riences a deshielding effect, whereas the acceptornucleus chemical shift moves to a lower fre-quency due to an overall increase in electronicshielding. Examples for these effects are donorand acceptor 15N chemical shift changes on H-

! .bond formation in nucleotides 102 , nucleic acid! .oligomers 103!105 , and histidine side chains

! .106, 107 . Similarly, ab initio calculations indi-cate that backbone H-bond formation in proteinsleads to a deshielding of the amide 15N nucleuswith a corresponding shift to higher frequencies

! .by approximately 13 ppm 108 .

Primary and Secondary Isotope Shifts fromSubstitutions of 1H by 2H and 3H

!The substitution of one isotope for another e.g.,2 1 .H for H is known to affect the isotropic chemi-

!cal shift of the substituted nucleus primary iso-. 13tope shift and of magnetic nuclei, such as C

and 15N, one or more covalent bonds away from! .the substituted nucleus secondary isotope shifts .

Whereas the primary isotope shift for the substi-tution of hydrogen by either deuterium or tritium

! .is usually small in weak H-bonds 109 , significant!effects are observed for strong H-bonds 110,

.111 . Such primary isotope shifts have been usedto characterize a number of H-bond systems andprovide information on the shape and symmetryof potential energy wells for the H-bonded hydro-

! .gen nucleus 14, 110!113 . Secondary isotopeeffects have also been used to characterize thepresence and properties of the weak H-bonds in

! .biomacromolecules 109, 114 .

Equilibrium 2H /////1H Isotope FractionationFactor

The 2 H!1H fractionation factor, %, is a quantitywhich is defined as the relative occupancy ofdeuterons versus protons at an exchangeable hy-drogen position of a solute, as compared with thecorresponding relative concentration of labile hy-drogens within a solvent of mixed proton anddeuteron content, e.g., 1H O!2 H O:2 2

$ 2 % $ 1 % $2 % $1 %% % D " H ! D " H ! H ! H! . ! .solute solvent

$ %1

Thus, a % value of less than one correspondsto an enrichment of protium relative to solventcontent, whereas a % value greater than unityreflects a preference for deuterium relative tosolvent content.

Experimental results suggest a correlation be-tween the fractionation factor % for an H-bondedhydrogen and the strength of the H-bond, wherea lower % value corresponds to a stronger H-bond. Although % is not an NMR parameter,NMR usually provides the most suitable methodfor its determination in small chemical molecules! .14 and biomacromolecules. % values for pro-

! . !teins 115, 116 vary over a wide range e.g.,! ..0.3!1.5 in staphylococcal nuclease 117 , and

have provided insights into the presence andstrength of H-bonds in secondary structures! . ! .117!120 and catalytic processes 121!126 .

Covalent Scalar Couplings

As scalar couplings are mediated by the electrons! .within chemical bonds see below , the redistribu-

tion of electron densities upon H-bond formationalso gives rise to observable changes in scalarcouplings between nuclei associated with the H-bond. Changes of one-bond couplings betweenhydrogen bonded proton and donor nuclei havebeen observed in a number of small chemicalcompounds in various organic solvents, wherechanges in 1J and 1J couplings were mea-NH CH

! .sured upon H-bond formation 127!130 . In par-ticular, for an adenosine!uridine base pair ana-logue in chloroform, base pair formation resultedin an increase of the imino group 1J fromNH

! ."91.3 to "87.5 Hz 131 . A similar observationhas been made for the 1J couplings of iminoNHgroups in different nucleic acid base pairs of a

! .DNA triplex 59 . This study also suggested thatan increase in the 1J coupling was associatedNH


with a decrease in the donor!acceptor distanceand thus with an increase in the ‘‘strength’’ of theH-bond. In proteins, H-bonding to the carbonyl

! .oxygen atom O C leads to a strengthening inthe sequential one-bond 1J coupling constantC "Nwithin the peptide bond, whereas H-bonding ofthe amide hydrogen results in a weakening of the1 ! .J coupling 132, 133 . This observation wasC "Nused to correlate the measured 1J couplingC "Nconstants with the various types of secondarystructure elements in a protein.

2H Quadrupolar Coupling Constant

! .Since the quadrupole coupling constant QCC isproportional to the size of the electric field gradi-ent at the position of the nucleus, the QCC of adeuteron within an H-bond is very sensitive to theasymmetry of the charge distribution within theH-bond. A shorter H-bond usually corresponds toan increased symmetry and thus results in weakerelectric field gradients and smaller 2 H QCC val-ues. In proteins, 2 H QCC values have been de-rived from the relaxation times of the deuterons

! .within H-bonds 134, 135 . In particular for pro-

. . .tein backbone N H O C H-bonds, a corre-lation to the H-bond length R and theH $ $ $ O. . . ! .N H O H-bond angle & & $ 120' could

!2 .be established as QCC H !kHz & 228 '3 ! .130 cos &!R 135 .H $ $ $ O

HYDROGEN BOND SCALAR COUPLINGS

Basic Principles of Scalar Coupling

Before discussing the observation in biomacro-molecules of scalar couplings between nuclei se-parated by a hydrogen bond, let us briefly discussthe characteristics of the principal interactionsbetween two magnetic nuclei in any substance,i.e., of the dipolar and the scalar couplings. Thedipolar interaction is transmitted through spaceby the magnetic dipolar fields surrounding thenuclei. Although this dipolar interaction can beon the order of tens of kilohertz, in isotropicsolutions and gases, the dipolar interaction aver-ages exactly to zero due to the isotropic reorien-tation of the molecules. In contrast to the dipolarinteraction, the scalar interaction is transmittedvia the electron cloud of the molecules. There-fore, this interaction is only observed betweennuclei that have electrons between them, i.e.,which are connected by some kind of chemical

bond. The scalar interaction is also called J-cou-pling, and is typically detected up to a distance ofthree to four bonds. In solution and gas phases,the J-couplings give rise to the familiar resonancesplittings that can be used to decipher the chemi-cal structure of molecules. The strength of thescalar interaction is measured by the size of the

! .resonance splitting in hertz Hz . This splittingdefines the scalar coupling constant, nJ , in which,i jn designates the number of bonds separating thetwo nuclei i and j. The value of the J-couplingconstant can be either positive or negative.

Let us describe the mechanism of scalar inter-actions between two nuclei A and X in more

!detail. If nucleus A is a spin-1!2 nucleus e.g.,1 .H , then there are two possible states for the z

! .component of its spin vector A & A , A , Ax y zwhen A is placed into a static magnetic field

! .B & 0, 0, B which is directed along the z axis.0 0These states are frequently referred to as the &and ( or ‘‘spin-up’’ and ‘‘spin-down’’ states. Alter-natively, the two states can be labeled by thequantum mechanical expectation values for A z! .'1!2 and "1!2 which is usually called themagnetic quantum number m . The magneticAmoment of the nuclear spin is proportional to the

A ! A A A .spin vector and is given as ! & ) , ) , ) &x y z*+ A, where + is the gyromagnetic ratio. TheA Ainteraction of the magnetic moment with a mag-netic field is called Zeeman interaction and thecorresponding Hamiltonian is given by

H Zeeman & "!A $ B & "*+ A $ BA 0 A 0

$ %& "*+ A B 2A z 0

The fact that A can have two different valuesz! .'1!2 and "1!2 leads to two different energylevels which are separated by an energy gap , E& *+ B . In nuclear magnetic resonance, we ob-A 0serve electromagnetic transitions between thesestates corresponding to electromagnetic radiation

! .with the resonance Larmor frequency - &A+ B . For a single nuclear species A, we detect aA 0single resonance line at position - . Note thatAfor a real chemical substance this picture is slightlyincorrect: the nucleus A is surrounded by elec-trons which slightly shield the external magneticfield. Therefore the resonance frequency - isAshifted by a tiny relative amount which is calledthe chemical shift.

The description of the interaction with themagnetic field is identical for the second nucleusX which for the sake of simplicity is also assumed


to be a spin-1!2 nucleus. In the absence of anyinteraction between the two nuclei, the totalHamilitonian of the two spin system is simply thesum of the two Hamiltonians:

Zeeman $ %H & "*+ A B " *+ X B 3AX A z 0 X z 0

As the two spins are independent of eachother, the energy levels for one spin do not de-pend on the state of the other spin and onesimply observes two independent electromagnetictransitions with Larmor frequencies - and - .A X

The situation changes when there is an inter-action between the two nuclei. Then the energylevels of one nucleus do depend on the state ofthe second nucleus and the total Hamiltonian forsuch a system contains an additional term whichdescribes the cross-talk between the two nuclei.Scalar interactions are caused by a two-stepmechanism. First, the magnetic moment of onenucleus magnetically polarizes the molecularelectron cloud. Then, the induced magnetic fieldof the electron cloud interacts with the magneticmoment of the second nucleus. As a conse-quence, the scalar interaction must be propor-tional to the magnetic moments of both nuclei Aand X. The most general Hamiltonian for such aninteraction is of the form

H J & !A $ K $ !X& )A K )X"AX & &( (& , (&x , y , z

2 $ %& * + + A K X 4"A X & &( (& , (&x , y , z

where K is a 3x3 matrix with elements K . The&(

fact that K is not a simple constant, but a matrix,expresses the possibility that the nucleus !electron ! nucleus polarization transfer mightdepend on the orientation of the molecule. How-ever, in the isotropic situation of liquids and gasesthe orientation dependent parts of K average&(

$ %to zero. As a result, Eq. 4 can be replaced by

H J & *2 + + K A X"AX A X & &&&x , y , z

2 $ %& * + + K A $ X % 2#*JA $ X 5A X

where the constant K represents the orientationindependent part of K , i.e., its trace, and the&(

scalar coupling constant J is given as$ %*+ + K!2 #. Equation 5 has the practical con-A X

sequence that, neglecting small additional iso-topic effects, the replacement of one nucleus byan isotopic nucleus results in a scaling of the

scalar interactions by the ratio of the respectivegyromagnetic constants. For example, the one-

13 2bond scalar coupling in a C H bond is usu-!1 . !2 .ally about a factor of 6.5 ( + H !+ H smaller

13 1than the corresponding coupling in a C Hbond.

$ %A further simplification for Eq. 5 can befound when the difference in the Larmor fre-quencies for nucleus A and X is large compared

& &to the scalar coupling constant J; i.e., - " -A X! 2# J. This limit is called weak coupling. In thiscase, the transverse parts of the scalar couplingHamiltonian which do not commute with theZeeman Hamiltonian are neglected:

J , weak $ %H & 2#*JA X 6AX z z

A pictorial explanation for this approximationcan be given by the fact that the transverse x andy components of both spins rotate very quicklyagainst each other in the external field when thedifference in Larmor frequencies is large. There-fore the transverse components of the scalar cou-pling quickly average to zero.

In any case, we see from the form of the scalar$ % $ %coupling in Eq. 5 or 6 that the energy levels of

one nucleus are influenced by the state of thesecond nucleus. If we assume that the z compo-nents of the nuclear spins A and X are parallel! .m & m & 1!2 or m & m & "1!2 , thenX A X Aaccording to the weak coupling Hamiltonian, theenergy of the system will be increased by theamount #*J!2. If both spins are antiparallel! .m & "m & 1!2 or m & "m & "1!2 ,X A X Athe energy will be lowered by the same amount. Aslightly more complicated description needs to be

$ %applied for the strong coupling case in Eq. 5 .There is yet another way to look at this: if we

assume that nucleus X is in a state where the zcomponent of its spin is given by m then we canX

!express the total Hamiltonian for spin A weak.coupling as

H total & "*+ A B ' 2#*JA mA A z 0 z x

! . $ %& " B " 2# Jm !+ + *A 70 X A A z

This means that the action of nucleus X onnucleus A looks like an additional magnetic fieldalong the z axis of size "2# Jm !+ at theX Aposition of A. Apparently this field corresponds tothe magnetic field that was induced in the elec-tron cloud by the magnetic field produced by spinX. The action of this induced field makes thenuclear spin A rotate faster or slower depending


on the sign of the J-coupling and the direction ofthe z component of the spin X. At thermal equi-librium in a solution containing many AX spinsystems, the two states m & 1!2 and "1!2 ofXthe spin X are almost equally populated. There-fore, the resonance line for nucleus A is split intotwo lines of identical intensity which are sepa-rated by the angular frequency difference 2# J. Inthe analogous situation where the frequency ofthe X nucleus is detected, an identical splittingof the spectral line is observed. From this descrip-tion of the scalar coupling effect it is apparentthat it leads to a correlation in the movement ofthe two interacting spins. The most fruitful use ofthis correlation is made in the transmission ofnuclear frequency information along chemical

! .bonds in COSY correlation spectroscopy experi-ments.

Electronic Interactions and the Transfer ofMagnetization across the Hydrogen Bond

So far, we have considered the size of the scalarcoupling constant as a phenomenological parame-ter. A theoretical determination of the value of Jinvolves the quantum-mechanical description ofthe electronic orbitals in a substance. Originallysuch a formulation of the spin!spin coupling was

! .put forward by Ramsey 136 who identified threebasic mechanisms which give rise to scalar inter-

! .actions, all mediated by valence electrons. 1 Thenuclear magnetic moments interact via the mag-netic field produced by the orbital motion of the

! . ! .surrounding electrons electron orbital term . 2The nuclear magnetic moments interact via themagnetic field produced by the spins of the sur-

! . ! .rounding electrons electron spin term . 3 Due tothe finite size of the nucleus, the nuclear mag-netic moments interact directly with electronsthat have a non-zero-probability density at the

! .position of the nucleus Fermi contact term . Inhydrogen-like atomic orbitals, only s-electronsfulfill this condition. The scalar coupling constantis given by the sum of all three terms. However,for couplings involving the hydrogen atom, suchas the H-bond scalar couplings, the Fermi contactterm is dominant and the other two terms can

! .usually be neglected 59, 81 . With the currentincrease of available computational power andthe advent of reliable numerical descriptions forthe electronic orbitals of non-trivial chemical

! .compounds such as GAUSSIAN 137 , it hasbecome feasible to carry out accurate calculationsof coupling constants based on these first princi-ples of electrodynamics for systems containing

several tens of atoms and a few hundred wavefunctions. For example, very good agreement be-tween experimentally determined H-bond scalarcoupling constants and theoretical simulationswere found for hydrogen bonds in nucleic acid

!base pairs and in the backbone of proteins 59,.81 . In these and other systems, such calculations

have become a very powerful analytical tool thatcomplements the experimental measurement ofscalar couplings.

Using this theoretical framework, we mightvisualize the transmission of nuclear polarizationacross the hydrogen bond between a donor nu-cleus D and an acceptor nucleus A in the follow-

! .ing way Fig. 1 : the nuclear magnetic moment ofthe nucleus D gives rise to a magnetic field whichorients an electron in its vicinity. The polarizationof this electron is transmitted via electron!elec-tron interactions, i.e., electric forces and the Pauliexclusion principle, to a second electron in thevicinity of nucleus A. The magnetic field pro-duced by this second electron polarizes the mag-netic nucleus of the acceptor A. In this situation,

!h2 .a two-bond scalar coupling J is observedDA!between both nuclei. Note that for n-bond scalar

couplings between two nuclei i and j across H-! .bonds, Pervushin et al. 54 introduced the sym-

bol hn J where the superscript h should indicatei j.that one of the n bonds is actually an H-bond.

An important consequence follows from the ob-servation of such an H-bond coupling: as theFermi contact term is dominant in the transfermechanism one can conclude that electrons withnon-zero density at the location of the nuclei onboth sides of the H-bridge must be involved inthe transmission. This means that the movement

Figure 1 Schematic representation of the scalar cou-pling mechanism across H-bonds. A two-bond couplingof type h2J connects the nucleus of the donor atomDAD with the nucleus of the acceptor atom A. The

! .electrons e transmit the magnetic polarization be-tween the two nuclei.


! .of electrons with s-electron character on bothsides of the H-bond must be correlated.

Hydrogen Bond Scalar Couplings inNucleic Acids

A Quantitati)e Trace of Unexpected MagnetizationLosses. Although a large body of observationsexisted about couplings between nuclei that werenot connected by usual covalent bonds, e.g., the

! .so-called ‘‘through space’’ couplings 138 or even!some observations of cross H-bond couplings 51,

.52 , until the late 1990s the common idea about ascalar coupling in a biological macromolecule wasassociated with a covalent bond. This is easilyexplained because the common H-bond couplingsin 13C and 15N isotope labeled proteins are rela-tively small, i.e., "# 1 Hz, and the observationof the larger H-bond couplings in nucleic acids! .up to # 11 Hz depended on the availability of15N labeled nucleic acids. Efficient labelingstrategies for the latter and the concomitant ap-plication of heteronuclear experiments becamewidespread only after such techniques had been

! .developed for proteins 139, 140 . In fact, ourobservation of such couplings in nucleic acidsresulted from the attempt to adapt heteronuclearprotein experiments for the use in nucleic acids.Unexpected losses of magnetization in such ex-periments revealed the presence of substantialcouplings across the H-bonds. Since the methodto detect such losses is very generally applicablefor the debugging of new pulse sequences, someof this evidence is presented here.

The original experiment was designed as anHNCO sequence where the imino 1H and 15N

! .resonances of uridine and guanosine Fig. 2should be correlated via a one-bond coupling tothe carbonyl 13C nuclei next to the imino 15Nnucleus, i.e., 13C4 or 13C2 in uridine and 13C6 inguanosine. Experience with proteins shows thatsuch an experiment is usually very sensitive, be-cause only relatively large scalar couplings areused in the magnetization transfer pathways. Typ-ical values of the imino 1J couplings are aroundNH"90 Hz, whereas typical 1J couplings rangeNC "

from "10 to "15 Hz. This means that the total!time for an INEPT transfer Insensitive Nuclei

! ..Enhanced by Polarization Transfer 141 be-tween the imino 1H and the 15N nuclei should be

$ ! 1 .%set to values of about 5.5 ms 1! 2 J and toNH$ ! 1 .%values of about 33 ms 1! 2 J for the trans-NC "

fer between the 15N nucleus and the adjacent

Figure 2 Hydrogen bond configuration and nomencla-! . ! .ture for A Watson!Crick U!A, B Watson!Crick! .G!C, and C U!G wobble base pairs. Approximate

! . 15isotropic chemical shift values in ppm for N nucleiare indicated. Dark shaded nuclei are involved in h2JNNscalar couplings.

13C-carbonyl nucleus. Neglecting numerical fac-tors and the chemical shift evolution periods, themagnetization path in such a HNCO experimentis given by

5.5 ms 33 ms '" "

H H N H N Cy z y z z y

33 ms 5.5 ms" " $ %H N H 8y z x

During this sequence, magnetization evolvesfor 11 ms as transverse proton terms and 66 ms astransverse nitrogen terms. Therefore, relaxation

!losses can be described by a factor exp "11!1 .. ! !15 .. !1 .ms!T H * exp "66 ms!T N , where T H2 2 2!15 .and T N are the relaxation times for the trans-2


1 15 $ %verse H and N magnetization terms in Eq. 8 .These relaxation times can be determined in in-dependent experiments. Assuming that the effi-ciency of the scalar magnetization transfer is closeto 1 and that the losses due to pulse imperfec-tions are small, the expected signal intensity ofthe whole experiment can be estimated from thesignal intensity in a simple one-dimensional pro-ton spectrum multiplied by the relaxation lossfactor. It turned out that the signal intensity ofthe HNCO experiment was much lower than ex-pected.

In order to determine the source of such amagnetization loss, it is often a practical way tobreak down a complete pulse sequence into singleparts that still lead to observable magnetization.

! .One such part is depicted in Fig. 3 A . It is an1H!15N heteronuclear single-quantum correla-

! .tion HSQC pulse sequence where the nitrogenevolution period has been replaced by the se-

( !15 .quence ,!180 N !,. It is recognized that theysequence would yield identical relaxation lossesas the HNCO sequence if the delay , is set to avalue of 33 ms.

14 13 12 11 10

x 0.25

U-A G-C G-U

Imino H1ppm

A)

B)

C)

D)

90 x 90 y180 y

δ δ

180 y

90 x 180 y

δ δ

DecoupleΔ Δ

90 x180 y 90 x 180 y

Preparation Evolution Refocusing Acquisition

N15

H1

Δ = 1 ms

Δ = 30 ms

Δ = 30 ms

Δ = 30 ms

Δ Δ ΔΔ

210 ppm

Δ Δ

Sel. 180

Non-sel. 180

ΔΔ

ΔΔ

152 ppm

152 ppm

152 ppm

152 ppm

a b c d

2 2 2 2

Figure 3 Original evidence of the h2J H-bond coupling effect in Watson!Crick baseNNpairs. Spectra were recorded at 25 'C on a 600 MHz Bruker DMX spectrometer using a

15 13 ! .1.6 mM sample of uniformly N! C-labeled PSTVd T1 RNA domain 69 nucleotides ,100 mM NaCl, 10 mM sodium phosphate, 95% H O!5% D O, pH 6.0. 15N spin-echo2 2

1 15 !modifications of a H N HSQC pulse sequence and resulting imino proton spectra see.text . Pulse flip angles and phases are indicated above each pulse. Unless noted otherwise,

1 !1 . !15 . 1carrier positions are H O H and 152 ppm N . RF field strength of all non-selective H215 15 !and N pulses were 29 and 6.3 kHz, respectively. N-WALTZ decoupling + B !2# &N 2

. 131.25 kHz was applied during data acquisition. For clarity C decoupling pulses are notshown. The delay . was set to 2.25 ms. A total of 32 scans per spectrum was collected. The

15 ! . ! . ! .selective N 180' pulses applied during the 2, period in C and D have a G3 153amplitude profile corresponding to an excitation bandwidth of !20 ppm.


The sequence in Fig. 3 is easily understood interms of the product operator formalism! .142!144 . Neglecting relaxation losses, the ini-tial INEPT period transfers longitudinal protonmagnetization of type H present at point a intozantiphase nitrogen magnetization of type 2H Nz yat point b. If this type of magnetization is alsopresent at point c, the reverse INEPT step willtransfer this operator product into observableproton magnetization of the type H at point d.xOther types of magnetization at point c shouldnot lead to observable magnetization at the endof the pulse sequence. To a good approximation,this is true for the depicted pulse sequence; if afurther suppression of the unwanted magnetiza-tion pathways is necessary, additional phase cy-cling steps and pulse field gradients can be ap-plied.

The interval between point b and c can beused to probe the fate of the operator product2H N under different conditions. For example,z y

( !15 .inserting the ,!180 N !, sequence leads to aynitrogen spin-echo experiment with proton excita-tion and detection. The 15N 180( pulse in theymiddle of the nitrogen spin echo serves to refocusthe 15N chemical shift evolution, presented by a

!Hamiltonian of the form - N assuming a con-N z.vention where * & 1

180'N- N , - N ,yN z N z" " " $ %2H N 2H N 9z y z y

15 ( !The N 180 pulse also decouples or refo-y.cuses any kind of heteronuclear scalar coupling

to nuclei such as 13C or 1H, presented for exam-ple by a Hamiltonian of the type 2# J N X :NX z z

180'N2 # J N X , 2 # J N X ,yNX z z NX z z" " "

2H N 2H Nz y z y

$ %10

( !15 .In both cases, the 180 N pulse reverses theydirection of the nitrogen x-magnetization in themiddle of the echo delay, and as a consequenceall the magnetization refocuses at point c. There-fore neither chemical shift nor heteronuclearcouplings should modulate the intensity of theproton signal which is detected at point d.

Figure 3 shows the results of this experimentfor the base-paired imino proton resonances ofthe 69-nucleotide potato spindle tuber viroid! .PSTVd T1 domain. The experiment for a totalspin echo 2, delay of 2 ms is illustrated in Fig.

! .3 A . Clearly observable are resonances downfieldof about 12 ppm which correspond to the iminoprotons for the Watson!Crick uridine!adenosine! . ! .U!A and guanosine!cytidine G!C base pairs.Upfield of 12 ppm, we see imino proton reso-nances which belong to non-Watson!Crick base

! .pairs, such as the G!U base pairs in Fig. 2 C .!When the delay 2, is increased to 60 ms Fig.

! ..3 B , the imino resonances from Watson!Crickbase pairs are reduced in intensity to a pointwhere they are no longer detected. In contrast,the intensity of the non-Watson!Crick G!Uimino resonances is attenuated to approximately25% such that these protons are still clearlyvisible.

During the interval 2,, a considerable signalloss is expected from the relaxation of the 2H Nz ymagnetization term. An independent T experi-1/

ment using 15N spin-lock fields with a radio fre-quency strength of approximately 2 kHz revealedthat the 15N transverse relaxation time for mostimino groups is approximately 45 ms at the 298 Ktemperature used for the experiments in Fig. 3.Neglecting additional losses due to 1H longitudi-nal relaxation, this 15N transverse relaxation time

! .leads to an expected decay of exp "60!45 & 0.26for the 2H N magnetization term during the 60z yms interval. Such a loss is close to the reductionin signal which is observed for the non-Watson!Crick imino resonances. However, it cannot ex-plain the much stronger signal loss for the Wat-son!Crick imino groups, considering that bothtransverse and longitudinal relaxation ratesseemed to be rather uniform for all imino groupsin the respective relaxation experiments. Appar-ently, the additional reduction in signal for theWatson!Crick base pairs is caused by an anothermechanism which is neither relaxation nor aheteronuclear scalar coupling.

Such a mechanism could be a homonuclear! . 15scalar coupling J between the imino N nu-NN

cleus and another 15N nucleus. If the radio fre-15 ! .quency strength of the N 180' pulse in Fig. 3 B

is comparable to or larger than the frequencyoffset of the 15N coupling partner, then thishomonuclear coupling would not be refocused. In

! .the case of the experiments of Figs. 3 A and! .3 B , an RF field strength0defined as the inverse

of the 360' pulse length0of 6.3 kHz was used.Therefore, such a 15N 180' pulse would effectivelyrecouple homonuclear 15N!15N couplings to part-ners within a range of approximately !100 ppm


! .B & 14 T around the carrier frequency. As-0suming that the 180' 15N pulse would affect bothnitrogen nuclei, the evolution under a homonu-clear coupling during the interval 2, is describedby

' 180'N , 180'N'2# J N N , y yNN" z z " "

2H Nz y

2# J N N' ,NN" z z " ! .2H N cos 2# J ,z y NN

' ! ." 4H N N sin 2# J ,z x z NN

$ %11

where N" represents the 15N nucleus of the cou-pling partner. Only the first of the two terms atthe end of the spin-echo period is transferred intoobservable magnetization by the reverse INEPTstep between time points c and d. Apparently, asignal resulting from this term will be modulated

! .by the factor cos 2# J , .NN! .The spin-echo time of 60 ms in Fig. 3 B re-

sults in a near complete loss of magnetization forthe Watson!Crick imino groups. If a homonu-clear coupling is responsible for this loss, then itwould be due to the first zero crossing of the

! .cos 2# J , factor, because no other zero cross-NNings were observed at shorter delay times. There-fore, such a coupling must have an approximate

! .strength of 1! 2"60 ms ( 8 Hz.To test this hypothesis further, the non-selec-

15 ! ! ..tive N 180' pulse Fig. 3 B was replaced by a15 ! ! ..selective N 180' pulse Fig. 3 C with an ap-

proximate excitation range of 152 ! 20 ppm thatcovers the frequencies of the guanosine and uri-

15 ! ! ..dine imino N resonances Fig. 2 A,B . Thisselective 15N 180' pulse not only refocuses anyheteronuclear scalar couplings, but also refocuseshomonuclear scalar couplings to 15N nuclei whichare outside of the excitation window of the pulse.Apparently, the application of the selective pulse

! .for a spin-echo delay of 60 ms in Fig. 3 C resultsin the recovery of the magnetization for the Wat-son!Crick resonances such that their intensitiesare equal to the intensities of the non-Watson!Crick G!U base pairs. This finding unambigu-ously establishes that the loss of magnetizationobserved with the non-selective 15N 180' pulse in

! . 15 15Fig. 3 B is caused by a homonuclear N! Nscalar coupling.

To further validate this result and to deter-mine the frequencies of the 15N coupling part-

ners, the non-selective 15N 180' pulse was placedback into the center of the spin echo delay. How-

!ever, two further selective decoupling pulses 210.! 20 ppm were applied in the middle of the two! ! .., delays Fig. 3 D . This has the effect that

homonuclear couplings to nuclei in this frequencyrange would be refocused at the end of the firstand second , delay. A similar recovery of magne-

! ! ..tization is observed Fig. 3 D as for the single! ! ..selective pulse centered at 152 ppm Fig. 3 C .

Therefore, it is obvious that the coupling partnersmust have resonance frequencies in the range of210 ! 20 ppm.

A Direct Obser)ation of the 15N Splitting. At thispoint, one may ask whether it is not possible toobserve the couplings directly as a splitting of the15N resonances. Of course, it is, but this is not asentertaining as doing the Fourier transform of

! .cos 2# J , in one’s head. The splitting can beNN1 15detected using a 2D H N HSQC experiment

where the 15N evolution period is made suffi-ciently long, i.e., significantly longer than 60 ms,such that the J coupling can be resolved. SuchNNan HSQC sequence is similar to the pulse schemeillustrated in Fig. 3 with the spin-echo delay beingreplaced by the familiar t -evolution period with a11H 180' pulse at its center. This proton pulserefocuses the 1J -coupling during the evolutionNHperiod. Similarly, couplings to the 13C nuclei ofthe 13C!15N-labelled RNA are removed by ap-propriate 13C 180' pulses. Since the followingarguments do not depend on any effects of cou-plings to 13C, these pulses are not shown in thefigures and the description of such effects hasbeen left out for clarity.

The product operator description during the$ %t -evolution is then very similar to Eq. 11 , except1

that the chemical shift precession is not refocused! . 15- t N , and thus N frequency labeling isN 1 zachieved:

2H Nz y

- t N '2 # J t N N'N 1 z NN 1 z z " ! . ! .2H N cos # J t cos - tz y NN 1 N 1

' ! . ! ."4H N N sin # J t cos - tz x z NN 1 N 1

! . ! ."2H N cos # J t sin - tz x NN 1 N 1

' ! . ! ."4H N N sin # J t sin - tz y z NN 1 N 1

$ %12


$ %Only the first term in Eq. 12 converges toobservable magnetization at the end of the pulsesequence. We note that for quadrature detectionin the t -domain, usually a sine-modulated term1for frequency labeling needs to be recorded. Thisis easily done by recording a second FID wherethe phase of the first 15N 90' pulse is changed

! .from x to y 145 . Complex Fourier transform ofthe t -dimension in such an HSQC experiment1gives rise to resonances which are split due to theactive J coupling. Figure 4 depicts a part ofNNsuch a 15N highly-resolved 1H!15N HSQC experi-ment containing the uridine imino resonances forthe U!A base pairs in the T1 domain of PSTVd.As anticipated, these imino resonances show anin-phase splitting of approximately 8 Hz in the15N dimension which is due to the 15N!15N scalarcoupling. Although this experiment provides in-formation regarding the magnitude of the cou-pling, it does not identify the coupling partner.

Figure 4 Uridine imino region of a highly resolved 2D1 15H N HSQC spectrum recorded on the PSTVd T1

!15 .RNA domain. The data matrix consisted of 300* N!1 . !) 1024* H data points where n* refers to complex

. !15 .points with acquisition times of 150 ms N and 77!1 .ms H . Total experimental time was 14 h. Other

experimental conditions as in Fig. 3. Resonances arelabeled with assignment information. Each uridineimino 15N3 resonance is split according to a h2JNNcoupling of # 8 Hz which correlates the uridine 15N3and the adenosine 15N1 nuclei through the H-bonds inthe respective U!A Watson!Crick base pairs.

Simultaneous Detection of Coupling Partners andQuantification of J Couplings—The Quantitati)eN NHNN-COSY. Both the chemical shift of the cou-pling partners and the magnitude of the nJNNcouplings can be determined effectively by usinga single quantitative, 15N-homonuclear COSY ex-periment. In this experiment, we can observe themagnetization which is transferred to the partner15 ! .N nucleus or nuclei which we could notobserve in the 2D 1H!15N HSQC approach de-scribed above. As the rate of magnetization trans-fer in COSY experiments is given by the magni-tude of the coupling constant, the intensity of thecross-peaks presents a measure for the size of the

! . ! .coupling 146 . Figure 5 A depicts the basicHNN-COSY pulse scheme employed to observeand quantify such nJ couplings in nucleic acidNNbase pairs. During the first INEPT part of thissequence, from time point a to time point b,magnetization is transferred from the imino pro-ton of one base via the covalent bond to theimino nitrogen nucleus to give the familiar an-tiphase magnetization term 2H N . During thez yfollowing first 15N!15N COSY delay from point bto point c, part of this magnetization is trans-ferred onto the 15N" coupling partner nucleus as

' ! .an operator term "4H N N sin 2# J , , whilez x z NNanother part remains on the imino 15N nucleus as

! .an operator term 2H N cos 2# J , , just asz y NN$ %described previously by Eq. 11 .

To ensure maximum excitation of both 15Nnuclei and thus transfer of magnetization via thescalar coupling, the frequency of the 15N 180(

ypulse in the middle of the COSY period is cen-tered approximately at the midpoint between thechemical shift of the imino nuclei and the chemi-

! .cal shift of the coupling partner i.e., # 185 ppm .Apart from its purpose to refocus the chemicalshift evolution of the imino 15N nucleus and torecouple the second 15N nucleus, this pulse alsodecouples the 15N imino nuclei from any he-teronuclear coupling partners. The following 15N90( COSY mixing pulse at time point c does notyaffect the nitrogen in-phase 2H N term, butz yrotates both nitrogen magnetizations in the nitro-gen anti-phase term "4H N N' :z x z

! . ' ! .2H N cos 2# J , " 4H N N sin 2# J ,z y NN z x z NN

90'N , 90'N'y y " ! .2H N cos 2# J ,z y NN

' ! . $ %' 4H N N sin 2# J , 13z z x NN

During the evolution period t , between time1points d and e, both 15N and 15N" nuclei precess


! . ! .Figure 5 A Basic HNN-COSY pulse sequence 53 . Narrow and wide pulses correspond1 !1 .to flip angles of 90' and 180', respectively. Carrier positions are H O H and 185 ppm2

!15 . 1 15N . All regular H and N pulses are applied at an RF field strength of 29 and 5.8 kHz,! . 1 ! .respectively. Low-power water flip-back 90' H pulses illustrated as smaller narrow pulses

are applied at a field strength of 200 Hz. For clarity 13C decoupling pulses are not shown.Delays: . & 2.25 ms, , & 15 ms, 1a & 2.5 ms, 1b & 0.25 ms, 1c & 2.25 ms, 1d & 0.5 ms.Unless indicated, the phases of all pulses are applied along the x axis. Gradients are

! .sine-bell shaped, with an absolute amplitude at their center and durations polarities of! . ! . ! . ! . ! . ! . ! .G & 2.5 ' , 2.1 " , 1.35 ' , 21.35 ' , 0.2 ' , 0.4 ' , and 0.101 ms ' .1,2,3,4,5,6,7

! .B Two-dimensional HNN-COSY spectrum recorded on the PSTVd T1 RNA domain. The!15 . !1 . !data matrix consisted of 250* N ) 1024* H data points where n* refers to complex

. !15 . !1 .points with acquisition times of 40 N and 77 ms H . Total experimental time was12.3 h. Other experimental conditions as in Fig. 3. Positive contours depict the diagonalresonances for the G and U imino groups. Negative contours correspond to cross peaksresulting from scalar 15N!15N magnetization transfer between the donor imino 15N nucleusand the acceptor 15N nucleus on the opposing base in Watson!Crick base pairs. Thediagonal peak marked by U7 represents a uridine imino group in a G!U wobble base pair! ! . .Fig. 2 C , see text . Resonances are labeled with assignment information. The insertillustrates the definition of the scalar h2J correlation via the H-bond.NN


according to their chemical shifts:

! . ' ! .2H N cos 2# J , ' 4H N N sin 2# J ,z y NN z z x NN

- N t '- N' tN z 1 N " z 1 " ! . ! .2H N cos 2# J , cos - tz y NN N 1

! . ! ."2H N cos 2# J , sin - tz x NN N 1

' ! . ! .'4H N N sin 2# J , cos - tz z x NN N" 1

' ! . ! .'4H N N sin 2# J , sin - tz z y NN N" 1

$ %14

Note that during the t -evolution, also the1homonuclear 15N!15N couplings and the het-eronuclear 1J coupling is active. Due to theirNHrelatively small size, the homonuclear 15N!15Ncouplings are not resolved during the maximalacquisition time for the t -evolution of approxi-1mately 40 ms. The effect of the heteronuclear1J coupling is used in the TROSY-type detec-NH

! .tion at the end of the pulse scheme see below .For a reverse-INEPT step at the end of the pulsesequence, a 1H 180' pulse would be necessary inthe middle of the t -period in order to remove the1splitting of the resonance line.

$ %Of the product terms in Eq. 14 , the secondand the fourth either do not refocus into observ-able magnetization at the end of the sequence orare suppressed by appropriate phase cycling. Thefate of the first and third terms after the second15N 90( COSY pulse at time point e and theyCOSY refocusing period until point f is the fol-lowing:

! . ! .2H N cos 2# J , cos - tz y NN N 1

90'N , 90'N'y y' "! . ! .'4H N N sin 2# J , cos - tz z x NN N" 1

! . ! .2H N cos 2# J , cos - tz y NN N 1

' ! . ! ."4H N N sin 2# J , cos - tz x z NN N" 1' 180'N , 180'N' '2# J N N , 2 # J N N ,y yNN " z z NN " z z" " "

2 ! . ! .2H N cos 2# J , cos - tz y NN N 1

' ! . ! . ! ."4H N N cos 2# J , sin 2# J , cos - tz x z NN NN N 1

' ! . ! . ! ."4H N N sin 2# J , cos 2# J , cos - tz x z NN NN N" 1

2 ! . ! ."2H N sin 2# J , cos - tz y NN N" 1

$ %15

As in the case of the normal HSQC, only thefirst and the last product operator terms, both oftype 2H N , are converted into observable iminoz yproton magnetization during the final TROSY! .147 part of the pulse sequence from point f topoint g. The TROSY-type detection scheme will

not be discussed any further here, except to pointout that a reverse-INEPT pulse train would alsolead to observable magnetization. However, it isadvantageous to use the TROSY scheme, becauseit only detects the narrow component of the1 15H N doublet, thereby increasing the sensi-tivity of the experiment for larger molecules. It is

$ %apparent from equation Eq. 15 that the ampli-tudes of the two 2H N terms contain the factorsz y

2 ! . ! . 2 ! .cos 2 # J , cos - t and "sin 2 # J ,NN N 1 NN! .cos - t . Fourier transform with respect to tN" 1 1

yields resonances in the f frequency domain at1position - and - . The intensities of theseN N"

diagonal and cross-peaks are proportional to2! . 2! .cos 2# J , and "sin 2# J , , respectively.NN NN

We also note that for quadrature detection usinga complex Fourier transform, it is necessary torecord magnetization terms which are pro-

2 ! . ! .portional to cos 2 # J , sin - t andN N N 12! . ! ."sin 2 # J , sin - t , respectively. TheseNN N " 1

sine-modulated terms can be obtained as observ-able magnetization at the end of the pulse se-quence if the phases of both 90' 15N-pulses atpositions b and c are rotated by 90'.

The result of such a quantitative HNN-COSYexperiment for the T1 domain of PSTVd is shown

! .in Fig. 5 B . For each Watson!Crick base pair weobserve a diagonal and a cross-peak. The diago-

! ! . .nal peaks Fig. 5 B , top correspond to the imino1 15 1 15H1 N1 and H3 N3 frequencies ofguanosine and uridine, respectively. The cross-

! ! . . 1peaks Fig. 5 B , bottom correlate the imino Hfrequencies to 15N resonances around 197 ppm inthe case of guanosine and to resonances around222 ppm in the case of uridine. To which 15Nnuclei do these cross-peaks belong? Based on the15N chemical shifts of the uridine cross peaks, wecan exclude the uridine 15N1 nuclei with typicalresonance frequencies of approximately 145 ppm! ! .. 15Fig. 2 A . The N1 nucleus would be the only

! 15 .other nitrogen nucleus besides N3 within thecovalent structure of uridine. Within the H-bonded structure of a Watson!Crick U!A base

! ! ..pair Fig. 2 A , the adenosine hydrogen bond N1acceptor atom is the next closest to the uridineimino N3 atom. The chemical shift of the adeno-sine 15N1 nucleus has typical values of approxi-mately 222 ppm. Therefore its chemical shiftmatches the cross peaks observed for the uridineimino resonances. Such an assignment can bevalidated by other experiments, e.g., a NOESYwhich connects the H3 proton of uridine and theH2 proton of adenosine and a long-range1 15 1H N HSQC which correlates the H2 and15 ! .N1 frequencies of adenosine 53 . Therefore,


we can conclude that we have identified a two-bond scalar coupling across the H-bond which isactive between the uridine 15N3 and the adeno-sine 15N1 nuclei in a Watson-Crick U!A basepair. Following the notation introduced earlier weterm this coupling h2 J .NN

The assignment of the guanosine cross peaks! .in Fig. 5 B is completely analogous. Within a

Watson-Crick G!C base pair, the hydrogen bondacceptor for the imino group of guanosine is theN3 atom of cytidine. The observed chemical shiftrange of 195!200 ppm for the guanosine cross-

! .peaks in Fig. 5 B matches closely to typicalchemical shifts of approximately 197 ppm for the15 ! ! ..N3 nuclei in cytidine Fig. 2 B . Therefore, alsoin the case of the G!C base pairs, the cross-peaks

1 15for the H1 N1 imino resonances of guano-sine correspond to a h2 J correlation with theNNH-bond cytidine 15N3 acceptor nucleus.

The magnitude of these h2 J couplings can beNN! .determined from the ratio of the cross-peak Ic

! .to diagonal peak I intensities observed in thed! $ %.HNN-COSY experiment Eq. 15 . The ratio of

these intensities is given by

2 ! h 2 . 2 ! h 2 .I !I & "sin 2# J , !cos 2# J ,c d NN NN

2 ! h 2 . $ %& "tan 2# J , 16NN

It should be kept in mind that only the abso-lute value of h2 J can be determined by thisNN

2 ! h2 .method, because both sin 2 # J , andNN2! h2 .cos 2# J , terms are not sensitive to a signNN

change of h2 J . A simple rearrangement ofNN$ %equation Eq. 16 allows the straightforward cal-

culation of this value:

1!2h 2& & ! . ! . $ %J & arctan "I !I ! 2#, 17! .NN c d

As calculated from the intensities of the! .HNN-COSY of Fig. 5 B , the absolute sizes of the

h2 J couplings are 7!8 Hz. These are in agree-NNment with the values measured from the line

1 15 ! .splitting observed in the H N HSQC Fig. 4and the estimate derived from the zero crossingof the intensities in Fig. 3.

Interestingly, the imino group of one uridine! . ! .U7 in Fig. 5 B has no H-bond correlation. This

! ! ..uridine is part of a U!G base pair Fig. 2 C . Forsuch a U!G base pair, the acceptor atom for theuridine imino proton is an oxygen atom ratherthan a nitrogen atom. Therefore no h2 J corre-NNlation can be seen in the HNN-COSY experi-ment. This returns us to the question posed ear-lier on why the magnetization for the

Watson!Crick H-bonded imino resonances de-! .cayed at different rates in Fig. 3 B as compared

to the resonances which are upfield of 12 ppm.The answer is straightforward: as all these iminogroups are part of non-Watson!Crick base pairswith oxygen acceptor atoms, the magnetizationdecays only by relaxation and not due to the15 15N N scalar coupling mechanism.

Other Types of H-Bond Couplings in Nucleic Acids.Besides the observation of h2 J couplings inNNWatson!Crick base pairs, H-bond scalar interac-tions between imino as well as amino 15N nucleiand the 15N nuclei of aromatic nitrogen H-bondacceptors have been observed in a number of

! .non-Watson!Crick base pairs 59, 61, 64, 65 . Inthe case for H-bond scalar interactions between15N amino donor and aromatic acceptor nuclei,the frequency separation between the amino and

15 ! .aromatic N resonances i.e., * 100 ppm is toolarge to be covered effectively by the limitedstrength of the currently available non-selective

!radio frequency pulses e.g., 6!7 kHz and a mag-.netic field strength of 14 Tesla . This problem is

! .overcome in the pseudo-heteronuclear H N N-COSY experiments where band-selective 15N

! . 15pulses 61, 65 excite the N amino donor andaromatic acceptor resonances separately. Furthermodifications of the HNN-COSY scheme havebeen proposed for the observation of h2 J corre-NNlations where the hydrogen nucleus in the hydro-gen bond is unobservable due to intermediateconformational exchange of hydrogen bondingamino groups or due to exchange of the proton

! .with the solvent 62, 66, 72 . In this situation,nearby carbon-bound protons can be used asstarting and end points for the magnetizationpathway. In almost all cases, values of h2 J areNNin the range of approximately 6!10 Hz, such thatthere is no indication of any fundamental differ-ence in the mechanism of magnetization transferacross H-bonds from either imino or amino groupsto aromatic nitrogen acceptors.

In addition to the h2 J couplings observed inNN. . .N H N systems, H-bond scalar couplings ofthe type h1J have also been observed in aHNnumber of base pairs where these one-bond cou-plings connect the imino protons to the 15N nu-

! .clei of nitrogen H-bond acceptors 54, 59, 76 .The size of these h1J couplings ranges betweenHN2 and 4 Hz. Due to their small size and the fasterrelaxation times of the imino proton, the detec-tion of the h1J couplings is more challengingHNthan for the h2 J couplings.NN


Apart from the aromatic nitrogen atoms, manynucleic acid base pairs involve oxygen atoms of

!carbonyl groups as hydrogen bond acceptors i.e.,

. . . .N H O C , for example, the G!U base! ! ..pair Fig. 2 C . Although the size of scalar cou-

plings across the hydrogen bond to the magneti-cally active oxygen isotope 17O is likely to be onthe same order as the couplings to a nitrogen 15Nacceptor, the fast relaxation of this quadrupolar

! .nucleus I & 5!2 would prevent the observationof such a coupling. Instead hydrogen bond cou-plings to the next possible nucleus of the carbonylacceptor group, the carbonyl 13C carbon, have

! .been observed in guanosine quartets 65 . The!h3 .size of the observed three-bond couplings JNC "

from the imino 15N donor nucleus to the carbonyl13 ! .C acceptor nucleus is approximately 0.2 Hz 65as measured by a long-range HNCO experiment! .see protein section below using selective car-

13 !h4 .bonyl C pulses. Four-bond couplings J ofNNapproximately 0.14 Hz have also been observed in

! .a similar G-quartet system 67 where the scalarcouplings connect the imino 15N1 nuclei in

15 . . . 15guanosine N1 H1 O6 C6 N1 hydro-gen bond networks. Thus, these h3J and h4JNC " NN

couplings are much smaller than the h2 J cou-NNplings that we described above. Apparently, thelarger number of intervening bonds and possiblyalso the deviations from a linear hydrogen bond

! .geometry 65 reduce the efficiency of magnetiza-tion transfer across the electron cloud.

Hydrogen Bond Scalar Couplings inProteins

. . .N H O C Hydrogen Bonds. The predomi-nant hydrogen bond in proteins connects thebackbone amide proton of one amino acid to thecarbonyl oxygen atom of a second amino acid! .Fig. 6, insert . Protein secondary structures aresynonymous with the description of the backbone

. . .N H O C hydrogen bond networks. The

. . . . . .canonical patterns are H O , O H ,i k i k. . . . . .H O , O H for anti-parallel (-i-2 k'2 i-2 k'2. . . . . . . . .sheets, H O , O H , H O ,i k i k'2 i'2 k'2. . . . . .O H for parallel (-sheets, O Hi'2 k'4 i i'4. . .for &-helices, and O H for 3 -helices wherei i'3 10H and O stand for the backbone amide protoni kand oxygen atoms of the hydrogen bondedresidues i and k, respectively.

174

175

176

9.4 8.48.68.89.09.29.6 ppm

13CÍ13 V5

H68 I44

L67 F4

I44 H68

L50 L43

E64 Q2

I3 L15

R42 V70

F45 K48

V70 R42

S57 P19

T7 K11

I23 R54

K6 L67

sL43

V17

I44

V5 I13

L69E18

F45

T55

K6

V5

s

s

s

s

s

s

i

i

1HN

15N H

13C'residue i residue j

O

13Cα Hα

13C'O

13Cα

15N H

h3JNC'

! .Figure 6 Selected region of the 2D H N CO spectrum recorded on a sample of 1.6 mMuniformly 13C!15N-labeled ubiquitin, 95% H O!5% D O, pH 4.6. The data matrix con-2 2

!13 . !1 . ! .sisted of 65* C" ) 512* H data points where n* refers to complex points with!13 . !1 .acquisition times of 39 C" and 53 ms H . Data were recorded at 45 'C on a 600 MHz

! .Bruker DMX spectrometer 12 h total experimental time . Cross-peaks marked as

. . . h3 15Res Res are due to J H-bond scalar couplings between the N nucleus of residuei j NiC"j13 ! .i and C" nucleus of residue j see insert for definition . Residue names marked by the

superscript ‘‘s’’ denote incompletely suppressed sequential correlations between the 15Nnucleus of residue i and 13C" nucleus of residue i " 1. This incomplete suppression of 1JNC"

correlations is a result of the variation in the size of 1J couplings in the range of "13 toNC"15 13 '! ."17 Hz 132 . The superscript ‘‘i’’ demarks intraresidue two-bond N C correlations.i i


As in the case of nucleic acid hydrogen bondsinvolving carbonyl groups as acceptors, the car-bonyl oxygen nucleus in proteins is not accessiblefor detection of scalar couplings across the H-bond. However, in an analogous way, h3J H-NC "

bond couplings can be detected involving the 13Cnucleus of the carbonyl moiety and the 15N nu-

! .cleus of the H-bond donor 55, 58 . The detec-h3 . . .tion of these J couplings in N H O CNC "

hydrogen bonds in proteins is achieved by astraightforward modification of the HNCO exper-

! .iment 148, 149 used for the sequential assign-ment of protein backbones. In the conventionalHNCO experiment magnetization follows an‘‘out’’ and ‘‘back’’ path according to 1H Ni !15Ni

!13C"i"1 !15Ni !1H Ni. In order to achieve thetransfer between the amide 15N nucleus of oneamino acid and the carbonyl 13C nucleus of thepreceding amino acid, the delays for the N i !y2N iC"i"1 INEPT step are usually set to valuesx z

! 1 .slightly shorter than 1! 2" J . With typicalNC "1J values in the range of 13 to 17 Hz, thisNC "

corresponds to transfer periods of approximately30 ms. In an HNCO experiment suitable for mag-netization transfer by h3J , the INEPT delaysNC "

1 ! .are set to 133 ms ( 2! J 55, 58 such thatNC "

the one-bond transfer from the amide 15N nu-! i . 13cleus of residue i N to the carbonyl C nucleusy! i i-1.of residue i-1 2N C" is approximately re-x z

! 2! 1 .focused i.e., sin # J T ( 0, where T &NC "15 13.0.133 s . However, transfer by N C" cou-

plings with coupling constant values different from15 Hz will still occur. As a consequence, theresulting HNCO spectrum does not contain the

131 15i i i"1normal H N cross-peaks, butC"cross-peaks resulting from eventual long-rangecorrelations, such as the h3J couplings be-NiC "jtween the amide 15N nucleus of residue i and thecarbonyl 13C nucleus of the hydrogen bonded

! .residue j Fig. 6, insert .Figure 6 shows the result of such a modified! .H N CO experiment where magnetization was

transferred by long-range J scalar interactionsNC "

between the amide 15N and carbonyl 13C nuclei inhuman ubiquitin. As only amide 1H N and car-bonyl 13C frequencies were detected in this two-dimensional version of the experiment, the JNC "

1 N 13interactions are apparent as H C" cross-peaks. Many of these correlations correspond toH-bond h3J interactions within the ubiquitinNC "

secondary structure. For example, two cross-peaksare visible between the backbone amide and car-bonyl groups of residues isoleucine 44 and histi-dine 68 which are part of an antiparallel (-sheet.A summary of these interactions is shown by thearrows in the secondary structure diagram ofubiquitin in Fig. 7. Quantification of the h3JNC "

coupling constants can be achieved by compari-

G35Q40D39P38P37I36

M1

Q2

L8

F4

I3

T7

K6

T9

V5 I13

T14

T12

L15

G10

K11

E16

V17

S20 P19 E18D21

T22

I23

E24

I30

V26

K27

N25

K29

A28

Q31

K33

D32

E34

L71

V70

L69

H68

L67T66

S65

E64

Q41

R42

L43

I44

F45

A46G47

K48

Q49

L50

Q62I61

K63

N60

COO–

E51

D52G53 T55 L56 S57 D58

Y59R54

α-helix β 3 β 4 β 5 β 1 β 2

Figure 7 Secondary structure topology of ubiquitin. Arrows mark the h3J correlationsNC"

which were observed in the long-range HNCO experiment of Fig. 6.


son of the cross-peak intensities from this experi-ment to the intensities of sequential amide!carbonyl correlations measured in a second refer-ence experiment. Typical values of h3J cou-NC "

!pling constants range from "0.2 to "0.9 Hz 55,.57, 58 . Although the absolute size of these cou-

plings is small, it is possible to trace out completehydrogen bond networks in smaller, non-

! .deuterated proteins ## 10 kDa by using thislong-range HNCO method. For larger proteins,the sensitivity of the long-range HNCO rapidlydecreases due to 15N transverse relaxation lossesduring the extended magnetization transfer peri-ods. An improvement in sensitivity can be ob-

! .tained by the TROSY approach 147 , deutera-tion, and the use of larger magnetic field strengths.Such an HNCO-TROSY experiment was success-ful in observing h3J couplings in the perdeuter-NC "

ated 30 kDa ribosome inactivating protein MAP30! .63 . Another source of improvement in sensitiv-ity to the long-range HNCO experiment has beenshown by quenching scalar coupling mediated re-laxation by using adiabatic or composite-pulse

! .decoupling on the aliphatic carbons 68, 69 . Inthis study, a 50% sensitivity enhancement overthe standard long-range HNCO experiment per-formed on a 12 kDa FK506 binding protein wasobserved.

In addition to the h3J couplings, H-bondNC "h2 ! .couplings of the type J 56, 73 and of theHC "

h3 ! .type J 74 have also been observed betweenHC &

the amide proton of the donor amino acid andthe 13C carbonyl or 13C & nuclei of the acceptingamino acid. With typical values in the range of0!1 Hz, these h2 J and h3J coupling con-HC " HC &

stants are similar in absolute size to the h3JNC "

coupling values.

Other Hydrogen Bonds. A number of H-bondscalar correlations have also been observed inproteins for H-bonds that are not of the predomi-

. . .nant N H O C type. For example, the ear-liest observations of H-bond couplings in proteins! .51, 52 involved scalar interactions betweenamide protons and the nucleus of a cysteine-coor-

!113 199 .dinated metal atom Cd or Hg in rubre-doxin. Apparently these J-couplings of up to 4 Hzwere mediated via the hydrogen bonds of type

. . .N H S Me from the backbone amides tothe S atoms of the cysteine residues. H-bonds of

. . .type N H N have also been detected byscalar couplings in a hydrogen bonded pair of

! .histidine imidazole rings in apomyoglobin 60 . Inthis case, h2 J couplings in the range of 8 to 11NNHz connect the protonated 15N22 nucleus of one

histidine to the unprotonated acceptor 15N22 nu-cleus of the second histidine. H-bond couplingsinvolving protein amide groups as donors andphosphate groups as acceptors have been de-scribed in a molecular complex of Ras p21 and

. . .! .GDP 75 . In these N H O P H-bonds,couplings involve either the amide 15N nucleus!h3 . !h2 .J or the amide proton J of the donorNP HPamino acid and 31 P nucleus of the acceptor phos-phate group. In complete analogy to the HNCOexperiment, the h3J correlations can be de-NC "

tected in an HNPO experiment, the only differ-ence being the replacement of the 13C carbonyl

31 ! . h3 h2pulses with P pulses 75 . With J and JNP HPcoupling constants of up to 5 Hz in size, thedetection of such correlations is particularly im-portant because long-range information on theposition of phosphorus in biomolecular com-plexes is very hard to obtain by traditional highresolution NMR methods.

Correlation of H-Bond Couplings andProton Chemical Shifts

Strong correlations of the isotropic chemical shiftof the H-bonded proton and the H-bond scalarcoupling constants have been observed for both

! . ! .nucleic acids 59 and proteins 55 . Irrespective

. . .of base pair type, in nucleic acid N H NH-bonds, both h2 J and 1J increase linearlyNN HNwith increasing imino proton chemical shifts. Asimilar correlation is observed in protein

. . . h3N H O C H-bonds, where J valuesNC "!h3 .decrease J " 0 linearly with increasingNC "

!1 N . 1 Namide proton H chemical shifts. Such Hdownfield shifts have been correlated with de-

! .creasing H-bond lengths 94!98 . Thus, the ob-served correlations between H-bond couplingconstants and proton chemical shifts indicate thatboth parameters are mainly determined by thespatial arrangement of the donor and acceptorgroups, and that shorter H-bond lengths corre-spond to proton downfield shifts and stronger! . h2 h3absolute size J and J coupling con-NN NC "

stants both in nucleic acids and proteins.

Dependence of H-Bond Couplings onH-Bond Lengths and Angles

The comparison of H-bond coupling constants tothe exact H-bond geometry is currently hamperedby the limited availability of very high resolutionstructures and by the limited precision of hydro-gen atom coordinates which are determined byX-ray diffraction. In particular, there are no X-ray


diffraction data available for most of the nucleicacids for which H-bond couplings have been mea-sured. Statistical analysis of a number of otherhigh resolution nucleic acid structures showed

. . .that the variation of the N1 N3 distances in! .Watson!Crick base pairs is limited 59 . In these

. . .DNA and RNA structures, the average N1 N3˚ ˚distances are 2.92 ! 0.05 A and 2.82 ! 0.05 A for

! .G!C and A!T A!U base pairs. The shorter

. . . ! .N1 N3 distances in A!T A!U base pairs ascompared to G!C base pairs coincide with anincrease in the value of the h2 J coupling con-NN

! .stants from approximately 6!7 Hz G!C to ap-! . ! .proximately 7!8 Hz A!T, A!U 53, 54, 59 .

! .Density functional theory simulations 150 in-dicate that the different chemical nature of thedonor and acceptor groups in Watson!Crick G!C

! .and A!T A!U and in Hoogsteen A $ T basepairs have a very limited influence on the h2 JNN. . .!coupling constants # 0.2 Hz, for N N dis-

˚.tances from 2.7 to 4.0 A . Therefore, observeddifferences in h2 J values for G!C, A!T, A!U,NNand A $ T base pairs should be largely due to thedifferences in donor!acceptor distances. Neglect-

. . .ing angular variations, it follows that at N N˚distances of 2.8 to 2.9 A, a change in the value of

h2 J by 1 Hz corresponds to a change inNN˚donor!acceptor distance of 0.07 ! 0.01 A. De-

creases of h2 J coupling constants by approxi-NNmately 1 Hz have been observed at the ends ofhelical stems and have been interpreted as acorresponding increase in the ensemble average

! .of the donor!acceptor distance 59 .

. . .Compared to the geometry of N H NH-bonds in nucleic acid base pairs, thereis far greater variation in the geometry of

. . .N H O C H-bonds in proteins. A statisti-cal analysis of a number of crystallographic struc-

! .tures 40 showed that the average values for

. . . . . . . . .N O and H O distances and for N H O

. . . ˚and H O C angles are 2.99 ! 0.14 A, 2.06 !˚0.16 A, 157 ! 11', 147 ! 9' in the case of &-

˚ ˚helices and 2.91 ! 0.14 A, 1.96 ! 0.16 A, 160 !10', 151 ! 12' in the case of (-sheet conforma-tions, respectively. Thus, the measured average

. . . . . . ˚N O and H O distances are about 0.1 Ashorter in (-sheets as compared to &-helices.This coincides with an increase in the averagestrength of the observed h3J coupling fromNC "

"0.38 ! 0.12 Hz for &-helical conformations to! ."0.65 ! 0.14 Hz for (-sheets in ubiquitin 55 .

An exponential correlation between the h3JNC ". . .coupling constant and the N O distance was! . h3described for protein G 57 where J isNC "

3 ˚$ %given as "59"10 Hz* exp "4"R !A . Ne-NO

glecting again angular dependencies, it followsfrom this relation that for a typical &-helical or(-sheet conformation, a variation in the value ofh3J by 0.1 Hz corresponds to a change in theNC ". . . ˚N O distance of 0.07 or 0.05 A, respectively.

Detailed information about the angular depen-dencies of the trans-hydrogen bond coupling con-stants is currently limited. Density functional the-

&h3 &ory simulations indicate that the J value hasNC ". . .a maximum for an N H O angle of 180' anddecreases in value by approximately 20 to 30%

! .for a decrease in this angle to 140' 81 . Therelation between the size of h3J and theNC ". . .H O C angle is less well understood. Arecent study showed that for similar H-bond dis-tances the h3J couplings in a nucleic acid G-NC "

tetrad were weaker than the h3J couplingsNC "! .observed in proteins 65 . The main difference in

the geometry of the two systems is that typical

. . .H O C angles in proteins have values ofapproximately 150' whereas the average

. . .H O C angle in the G-tetrad is 125 ! 5'.This points towards a similar angular dependency

. . .as for the N H O angle, namely, that a devi-

. . .ation from a straight H O C conformationyields weaker h3J couplings. Support for thisNC "

interpretation comes from very recent observa-h3 . . .tions of J couplings in N H O P H-NP

! . h3bonds 75 . In this study a J coupling of 4.6NP. . .Hz was observed for an H O P angle of 173',but the values of h3J dropped below 0.35 Hz forNP . . .conformations where the H O P angle wassmaller than 126'.

CONCLUSION

This review should serve as an introduction to thephenomenon of trans-H-bond scalar couplings innucleic acids and proteins. In particular, we fo-cused on the observation of h2 J couplings inNNWatson!Crick base pairs by illustrating how thecouplings were initially detected and character-ized. Since the H-bond scalar couplings follow thesame electron-mediated polarization mechanismas the covalent scalar couplings, the NMR con-cepts for their description and the experimentalprotocols for their detection are identical. Forexample, the detection of h3J couplings in nu-NC "

cleic acids and proteins can be achieved by astraightforward modification of the conventionalHNCO experiment. In contrast to the strong H-bond couplings involving nitrogen acceptor atomsin many of the nucleic acid base pairs, the size ofH-bond couplings involving oxygen acceptor


atoms, such as the h3J couplings in proteins, isNC "

typically only a fraction of a Hertz. Nevertheless,the use of carefully optimized experiments makesmany of these interactions detectable in small tomedium-sized biomacromolecules.

Clearly, most of the applications for the H-bondcouplings will be directed towards the unambigu-ous establishment of H-bond networks in thesecondary and tertiary structures of proteins andnucleic acids and their complexes. However, thesize of the H-bond scalar coupling could also beapplied to provide previously inaccessible infor-

! .mation in other cases; e.g., 1 as the H-bondcoupling constants are correlated to the H-bonddistance and angles, they could be used as a

! .precise measure for the H-bond geometry. 2Changes in the size of the coupling constantsshould provide an indication for tiny changes inH-bond geometry when biomacromolecules aresubjected to different physicochemical conditions,such as changes in temperature, pH, chemicaldenaturation, or ligand binding. Such repro-ducible changes could indeed be found and ratio-nalized by an induced fit mechanism in a study ofthe formation of a complex between an SH3

! .domain and a small peptide 151 . The observa-tion of H-bond couplings is also possible duringprotein folding. In this case the size of the cou-pling constants provides a quantitative measurefor the population of the closed and open statesof the hydrogen bonds. A recent application onthe TFE-induced helix formation of the RNase AS-peptide shows that such data can be interpretedin the context of common coil to helix transition

! . ! .theories 152 . 4 The coupling constants couldbe used to characterize H-bond networks in theactive centers of enzymes and ribozymes. In par-ticular, the proposed low-barrier H-bonds shouldgive rise to very strong coupling values.

ACKNOWLEDGMENTS

We thank Professors Juli Feigon and MichaelBarfield for very fruitful discussions, and Profes-sors Detlev Riesner and Georg Buldt for continu-öus support. A.J.D. is a recipient of an AustralianNational Health and Medical Research CouncilC.J. Martin Postdoctoral Fellowship. F.C. is arecipient of an A. v. Humboldt fellowship.

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Andrew J. Dingley completed his under-graduate studies at The University ofSydney in 1991, majoring in biochemistry.He continued with a Ph.D. under thesupervision of Glenn F. King developingtechniques to measure the translationaldiffusion coefficient of macromoleculesusing pulsed-field-gradient NMR. In 1997he joined the laboratory of Stephan

Grzesiek at the Forschungszentrum Julich as a postdoctoral¨fellow, where his research has focussed on the development ofheteronuclear, multidimensional NMR techniques for struc-tural studies of nucleic acids.

Florence Cordier completed her under-graduate studies in physics at the Univer-sity Joseph Fourier of Grenoble in 1994.In 1998, she obtained a Ph.D. formethodological developments in high res-olution NMR and dynamical studies ofproteins working in the laboratory of Do-minique Marion in Grenoble. Since thenshe has been a postdoctoral fellow in the

group of Stephan Grzesiek at the Forschungszentrum Julich,¨Germany, and at the Biozentrum Basel, Switzerland. Currentresearch interests include the interpretation of scalar cou-plings across hydrogen bonds in terms of hydrogen bondgeometry, the influence of physico!chemical parameters onthe hydrogen bond network in proteins, as well as generalstructural and dynamical properties of proteins in solution.

Stephan Grzesiek received his Ph.D. inphysics from the Free University of Berlinin 1988 for optical studies of proton re-lease in bacteriorhodopsin. During apostdoctoral stay at the Roche phara-maceutical company in Basel, Switzer-land, he was introduced to high resolu-tion NMR and continued from 1991 as avisiting fellow in the laboratory of Ad

Bax at the National Institutes of Health, USA. In 1996, he wasappointed to Professor for Biological NMR Spectroscopy atthe University of Dusseldorf, Germany. In 1999, he joined the¨Biozentrum of the University of Basel, where he is presently aprofessor in the Department of Structural Biology. His workfocuses on the development and application of NMR methodsfor the determination of structure and function of biologicalmacromolecules.

Documents

An introduction to hydrogen bond scalar couplings · not all, interactions involving biological macromolecules. For both proteins and nucleic acids, ... order of 100!600 kJ mol"1