NMR of Proteins - University at · PDF fileDetermining Protein Structures by NMR • the process of determining a solution structure by NMR is one of measuring many (hundreds/thousands)

NMR of ProteinsNMR of Proteins

Determining Protein Structures by NMRDetermining Protein Structures by NMR

• the process of determining a solution structure by NMR is one of measuring many (hundreds/thousands) of short proton- proton distances and angles, and restraining the protein structure with these computationally

H H ….}d

1H NMR Spectra of Proteins1H NMR Spectra of Proteins• 1D, 1H NMR spectra of even small proteins are impossible to interpret in any comprehensive manner -normally, only gross statements about secondary structure, tertiary structure, etc. can be made

ubiquitin (76 amino acids, 8.5 kDa)

simple 1D 1H experiment

90 90t1COSY

t2

2D 1H “COSY” experiment cytochrome c, 12.5 kDa

• for even moderate sized proteins, addition of a second dimension still does not alleviate spectral crowding and overlap in 1H spectra

nD, heteronuclear NMR Spectra of ProteinsnD, heteronuclear NMR Spectra of Proteins

• Modern NMR spectroscopic studies of proteins rely on multidimensional experiments involving 1H, 13C, and 15N nuclei in isotopically labeled proteins• These methods provide for signal selection (selectivity) and a means to reduce signal overlap


simple 2D 1H, 15N “HSQC” experiment

• In order to measure the distances between protons, we need to find out what protons give rise to the signals in the spectra, I.e. we have to “assign” the protein (figure out the chemical shifts for all of the protons)• The methods used are based on heteronuclear spectra

• applicable to uniformly isotopically enriched proteins -uniform 13C and 15N labeling: spin 1/2 -three nuclei (1H, 15N, and 13C) are involved

• based on magnetization transfer via (mostly) one bond J couplings -most of these couplings are large compared to linewidths for moderate sized proteins (~20 kDa) -magnetization transfer is efficient -indirect (1H) detection

• provides selective chemical shift correlation -spectral degeneracy minimized

Triple Resonance ApproachTriple Resonance Approach

• Proteins can be uniformly isotopically labeled by recombinant expression using defined media -bacterial expression most common

-also yeast, and cell-free systems are being developed -minimal media using 13C6 glucose as the sole carbon source and 15NH4Cl (or -SO4) as the sole nitrogen source

-normally >98% atom excess-also labeled “rich” media ($$)

-for larger proteins, uniform or fractional 2H labeling also used-2H, 13C glucose and D2O

Uniform Isotopic Labeling of ProteinsUniform Isotopic Labeling of Proteins

- these 1J and 2J couplings are uniform throughout polypeptides/proteins

- these 1J and 2J couplings are virtually conformation independent

1J and 2J Couplings in Proteins1J and 2J Couplings in Proteins

• correlates the chemical shifts of 1HN, 15N, 13Cα

i and 13Cαi-1

Prototypical Triple Resonance Experiment: HNCAPrototypical Triple Resonance Experiment: HNCA

• both 13Cαi and 13Cα

i-1 chemical shifts are correlated -the peak for the intra-residue correlation is usually more intense (11 Hz 1JNCα coupling vs 7 Hz 2JNCα coupling)

1H, 15N-HSQC HNCA

Prototypical Triple Resonance Experiment: HNCAPrototypical Triple Resonance Experiment: HNCA

2D HNCA projection 3D HNCA

Triple Resonance Approach: A Simple ExampleTriple Resonance Approach: A Simple Example


• link the correlated shifts numerically….

…. or visually


• problems:13Cα chemical shift degeneracy in proteins13Cα linewidths/resolution

-these preclude complete linkage via 13Cα alone -the same is true for 13Cβ, 13C′

Triple Resonance Approach: HNCA/HN(CO)CA ExampleTriple Resonance Approach: HNCA/HN(CO)CA Example

• The Nuclear Overhauser Effect or Nuclear Overhauser Enhancement is the change (enhancement) of the signal intensity from a given nucleus as a result of exciting or saturating the resonance frequency of another nucleus• It is based on through-space interactions• The magnitude of the effect is dependent on distance -enhancement depends on 1/r6, where r is the internuclear distance -thus, the effect is limited to distances of approximately 5Å or less

• This provides a means to determine if any two protons in a protein are < 5Å apart• The basic experiment used for proteins is called a NOESY

90 90 90

τmt12D NOESYt2

The Nuclear Overhauser EffectThe Nuclear Overhauser Effect

cytochrome c, 12.5 kDa

• Even for relatively small proteins, the 2D NOESY spectrum is hampered by severe spectral overlap

-pulse sequences: combine 2D sequences to get 3D sequences-get increased dimensionality and increased resolution without an increase in the number of signals (peaks)

90 90 90

τmt1NOESYt2

HSQC

τ

180 90

180 90

1H

15Ndecouple

90 180 180

18090

τ τ τ

t1/2 t1/2

90 t2

90 90

τmt1

NOESY-HSQC

τ

180 90

180 90

15Ndecouple

90 180 180

18090

τ τ τ

t2/2 t2/2

90 t31H

3D NOE Experiments for Distance Restraints3D NOE Experiments for Distance Restraints

Left: 2D NOESY

Far left: 2D plane of 3D NOESY- HMQC (1H, 13C)

-1H signals resolved by 13C chemical shifts of bound 13C atoms

3D NOE Experiments for Distance Restraints3D NOE Experiments for Distance Restraints

Structure CalculationsStructure Calculations• the primary structural restraint information for high resolution protein structures are the• NOE-based distance restraints -additional restraints include angle restraints based on coupling constants, long range restraints based on dipolar couplings, hydrogen bond restraints• calculation of structures involves satisfying structural restraints using simulated annealing/• restrained molecular dynamics -a target/energy function including terms for covalent geometry (known bond lengths and bond angles) and experimental restraints is minimized -the molecule is computationally heated/cooled to attempt to find a global minimum• the number of restraints is an important indicator of the quality of final structures

Practical Aspects of Protein StructureDetermination using NMR

Practical Aspects of Protein StructureDetermination using NMR

IntroductionIntroduction• NMR vs X-ray crystallography for protein structure determination

• in an x-ray diffraction pattern, each datum (reflection) contains information about each atom in the asymmetric unit -each atom contributes information that contributes to the intensity of each reflection• in an NMR spectrum, each datum (peak) contains information about only a single interatomic distance or angle -the process of determining a solution structure by NMR is one of measuring many small distances and angles “one at a time”

• Why use NMR?• can’t get a crystal / want to work in solution• want to look at binding to other proteins/molecules• want to understand stability• want to measure fast dynamics processes

• Protein production• Protein purity• Isotopic labeling• NMR samples / conditions / tubes• Simple spectra / evaluation (stability, tertiary structure)• Protein size / magnet size

IntroductionIntroduction

• Some things you should know before you talk to an NMR spectroscopist about determining a structure

Protein productionProtein production

• protein sample(s)• in theory, as little as a few mg of protein is sufficient

-best if the protein sample for NMR is > 1 mM• in practice, tens of milligrams (or more) are usually necessary, as are multiple samples -multiple samples if your protein is not stable -multiple samples with different isotopic labeling schemes• many very good bacterial expression vectors/cell strains are available for expression in bacteria

-good track record, easily automated• expression in eukaryotic cells more complicated (yeast, insect, human cells)• are cell-free systems currently available (i.e. Roche “Rapid Translation System (RTS))….good for specific isotope labeling, not good for uniform isotopic labeling

Protein purityProtein purity

• protein samples should always be as pure as possible• in practice, for small proteins, small amounts of high molecular weight contaminants are OK

SDS-PAGE

molecular weight markers sample

• protein must be uniformly isotopically labeled with 13C and 15N

• not difficult these days: bacterial expression cell strains grow well on minimal media (D-glucose (U-13C6, 98-99%) and 15NH4Cl (98-99%) as sole carbon and nitrogen sources, respectively)

• for 1L medium, $300 for glucose (2 g @ $150/g), and $30 for 15NH4Cl (1 g @ $30/g)

• there are also alternatives to minimal media (i.e. isotopically labeled rich media), but they are much more expensive

Isotopic labeling of protein for NMRIsotopic labeling of protein for NMR

• in practice, often multiple samples with other isotopic labeling schemes are necessary

• 15N only for certain angle (φ, ψ) measurements -also, usually used for initial evaluation of sample/spectra• 13C, 15N plus partial or uniform deuteration for large proteins -requires growth in minimal media in D2O -cells must be adapted to growth on D2O• samples with only specific amino acid types labeled assist in NMR resonance assignment -cells grown on medium with all unlabeled amino acids except for the one of interest -more common for larger proteins• samples made with a mixture of 10% U-13C6 glucose and 90% unlabeled glucose are used for stereospecific -CH3 assignment -proR methyl groups of Valine and Leucine



uniformly 15Nlabeled protein

15N-Gly onlylabeled protein

uniformly 13Clabeled protein

ε13CΗ3-Met onlylabeled protein

Left: sample prepared by growth on 100% uniformly 13C-labeled glucose

Right: sample prepared by growth on 10% uniformly 13C-labeled glucose and 90% unlabeled glucose

• isotopic labeling to identify specific amino acid types, groups, or to assign stereospecificity

• buffer: no C- or N-bound protons• phosphate is the best….• deuterated Tris, deuterated acetate, deuterated imidazole, etc., are OK (can be expensive)

• salts• K+, Na+, Cl-, SO4

2-, etc., all OK (no protons)• too much salt leads to decreased S/N

• pH: neutral or lower is best• must minimize the rate of exchange of amide protons with solvent

• solvent• 90% H2O, 10% D2O (for instrumental lock)

The NMR sampleThe NMR sample

The NMR sampleThe NMR sample• temperature

• sample dependent: usually 25 to 35 °C• bacteriostatic agents

• sodium azide used widely• put it all together in a good quality, clean NMR tube

• “standard” NMR tube is 5 mm diameter (for use in a 5 mm NMR probe)….volume of sample is ~500 - 700 uL (~1 mM protein)• magnetic susceptibility matched tubes, “Shigemi” tubes, permit lower volume samples to be used (i.e. less sample, or more concentrated sample), usually without deleterious effects (but the tubes are complicated)…volume of sample is 200-300 uL

Initial NMR spectra / evaluationInitial NMR spectra / evaluation

f (t) =1

2πF(ω)e−iωtdω

−x

x

∫

F(ω) = f (t)eiωtdt−x

x

∫

• 1D 1H NMR spectrum of a small organic compound

• 1D 1H NMR spectrum of a small protein

• for even small proteins, 1D spectra are complicated and cannot be analyzed comprehensively• 1D spectra can be useful, however, for evaluating the suitability/stability of a protein sample


acid-unfolded ubiquitin

ubiquitin, neutral pH


• 1D 1H NMR spectrum of a small protein

• for properly folded small proteins -peaks should be sharp -peaks should show good chemical shift dispersion (i.e. tertiary structure intact)

• for unfolded proteins -peaks are usually broad (many protons in each peak) -chemical shift dispersion poor (leading to the broad peaks)

• sample stability• can takes weeks of instrument time to acquire all data for structure determination• sample has to be stable for the amount of time necessary to acquire all of the data (at the data acquisition temperature), plus the time between experiments (all data is rarely acquired all at once)


protein x, t = 0

properly folded

protein x, t = 2 weeks(at room temperature)

partially unfolded

• simple 2D 1H, 15N correlation NMR spectra of proteins• reduce complex spectra to simple ones based on isotope editing• reduce/eliminate spectral overlap/spectral degeneracy• correlate amide 1H-15N pairs

• this spectrum demonstrates 1). that you can express your protein, 2). That you can isotopically label your protein, 3). That your protein is pure (1 peak per amino acid), 4) that your protein is folded (tertiary structure / good chemical shift dispersion), 5). etc.

• granting agencies need to see this spectrum or similar (akin to a crystal and a diffraction pattern)

simple 2D 1H, 15N “HSQC” experiment




protein x, t = 0properly folded

protein x, t = 2 weeks(at room temperature) partially unfolded

• tertiary structure and sample stability

c: apomyoglobinb and a: acid unfolded apomyoglobin

• chemical shift dispersion and peakwidths reflect tertiary structure

ubiquitin (76 amino acids, 8.5 kDa) AlgH (189 amino acids, 20.2 kDa) EPSP synthase (427 amino acids, 46.2 kDa)

Size (of the protein) mattersSize (of the protein) matters

• the rotational correlation time (τc) scales with protein size• larger τc: peak broadening and decreased S/N

• larger proteins have more atoms, therefore more peaks in the spectra• more peaks: increased peak overlap/chemical shift degeneracy

• 1H, 15N-HSQC (TROSY) spectra of EPSP synthase (46 kDa) at 600 and 800 MHz

• higher field means higher sensitivity (increased S/N), increased resolution (decreased peak overlap), and a bonus increase in S/N in TROSY experiments

600 MHz 800 MHz

Size (of the magnet) mattersSize (of the magnet) matters

NMR: beyond structureNMR: beyond structure

residue

S2

residue

• slow dynamics / local and global stability (hydrogen exchange)

• fast dynamics (ps/ns) via nuclear relaxation

• ligand binding

• mutational affects

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NMR of Proteins - University at · PDF fileDetermining Protein Structures by NMR • the process of determining a solution structure by NMR is one of measuring many (hundreds/thousands)