Andrea Buzády - PTE-TTK Fizikai Intézet · biomolecular function in vivo ⇔ ⇔ secondary and tertiary structure of bioplimers ⇔ dynamical motions in the relevant timescale at

„Preparation of the concerned sectors for educational and R&D activities related to the Hungarian ELI project ”

THz spectroscopy in biology

Andrea Buzády

5. lectureApplications

TÁMOP-4.1.1.C-12/1/KONV-2012-0005 projekt 1


5. lecture

Introductionbiomolecular function in vivo ⇔⇔ secondary and tertiary structure of bioplimers⇔ dynamical motions in the relevant timescale

at the cores of proteins – substituent amino acids: structure and function

directly monitor complex macromolecule dynamics in real timemotions of polipeptid chains at atomic levellow frequencies vibrations – terahertz regime

may be observed by: ♣ Raman spectroscopy♣ low-energy neutron spectroscopy♣ THz spectroscopy

Susan L. Dexheimer: Terahertz Spectroscopy Principles and Applications


5. lecture

Introduction

Susan L. Dexheimer: Terahertz Spectroscopy Principles and Applications

In case of: > 30 kDa molecular weigthsoverlapping states and frequencies of the collective modes →the absorption bands smear out, yielding structurless spectra

measurements in aqueous phase environments - because of biologic system

difficulties:- water absorption in 1- 3 THz region masking the absorption of biomolecules- spectral broadening: interconversion between numerous accessible conformational states

despite the limitation there are a lot of results- experimental- modeling method - simulations


5. lecture

Water is life – Water of Life – Water for life - …..Science of water at nanoscale

one oxigen and two hydrogen atoms – covalent bonds

apparent simplicity ⇔ anomalous properties (?)

We know the water: hydrogen-bonded bulk liquid melting at 0 °C and boiling at 100 °C

But this way the water may not exist within cells


5. lecture

Science of water at nanoscale

apparent simplicity ⇔ anomalous properties (?)

⇐ structure of molecules ⇐ binding to each other by hydrogen bonds

conventional picture of bulk water: tetrahedral motif

recently: questioned

http://www1.lsbu.ac.uk/water/


5. lecture


recently: in bulk water molecules bind on average to just two others

most molecules: arranged in strongly hydrogen bonded chains and in rings connected by weak hydrogen bonds

cluster network


P. Wernet, D. Nordlund, U. Bergmann, M. Cavallieri, H. Ogasawara, LA. Näslund, TK. Hirsch, L. Ojamäe, P. Gllatzel, LG. Pettersson, A. Nilsson: The structure of the first coordination shell in liquid water, Sciense, 304, 995-999 (2004)


5. lecture



M. Sonoda, N.H. Moreira, L. Mart´ınez, F.W. Favero,S.M. Vechi, L.R. Martins, and M. S. Skaf : A Review on the Dynamics of WaterBrazilian Journal of Physics, vol. 34, no. 1, March, 2004

Forms of water → In different molecular environment

Bulk water – only water molecules

Interfacial water –neighbouring molecules next to another media

Confined water – closing by molecules of another media


5. lecture

What are the properties of water in the biological environment?What are the properties of water in a living cell?

What are the properties of water in cytoplasmic packing density of up to 400 mg/ml of protein, nucleic acids, lipids, carbohydrates and small molecules or ionic compounds?

aquaporin protein - waterhttp://askabiologist.asu.edu/venom/protein-channels

http://www.exobiologie.fr/index.php/vulgarisation/chimie-vulgarisation/the-role-of-water-in-the-structure-and-function-of-biological-macromolecules/

Science of water and biomolecules


5. lecture

In cytoplasmic packing or in membran density of up to 400 mg/ml of protein, nucleic acids, lipids, carbohydrates and small molecules or ionic compounds?

there is little distance from any one molecule to its nearest neighbors –only about 20-30 Å

Roughly ten layers of water molecules can fit into these spaces different properties from water in “bulk” ⇐ interactions with cellular components

http://biologyct.blogfa.com/

just for demonstration



5. lecture

The time scales: PICOSECOND

♣ collective motions of solvent molecules

Modern terahertz instrumentation: possibility to observe water dynamics and the fast collective motion of water molecules, around biological molecules.

1012 Hertz = 1 Terahertz 1 picosecond

♣ collective, functionally important motions of large biomolecules such as proteins and nucleic acids.

♣ hydrogen bond rearrangement in water

coupled in the picosecondtime scale

http://www.hfsp.org/frontier-science/hfsp-success-stories/water-and-biological-molecules-probed-terahertz-spectroscopy



5. lecture

Science of water and biomoleculesD.M. Leitner, M. Gruebele, M. Havenith: Solvation dynamics of biomolecules: modeling and terahertz experiments, HFSP Journal 2, 6, 314–323 (2008)

complex macromolecules → great many vibrations → spectral density ρ (ω) - rather than vibrational peaks separately

modeling of five-helix bundle protein, ∗

> 90 THz: vibrations from the lightest nuclei (H)most localized vibrational modes ~ 30 THz: small amplitude stretching and bandingmodes of backbone and sidechains < 10 THz: delocalized large numbers of atoms1 – 5 THz: water absorption – significant rule

grey: proteinblack: water


5. lecture


Despite the „possibility” → trouble - little information contained in the THz spectrum of a biomolecule

BUT: measurements of the THz absorbance as a function of biomolecule concentration in solution→ can be deduced the extent of the hydration layer → the number of water molecules around the biomolecule

The frequency dependence of the absorbance is largely featureless in the 1-5 THz regimeBiomolecules absorb much less THz light than water or even: water absorb much more than biomolecule in terahertz regime

bulk water and hydration water - distinct absorption coefficients, identified to account for the variation of the absorbance with biomolecule concentration



5. lecture

experimental studies + molecular dynamics simulations modeling the same system


the change in absorbance with concentration

deduce the size of the hydration layer

the molecular level of the biomolecule-solvent dynamics underlying the THz spectra



5. lecture


♣ “Terahertz defect”: over part of the THz frequency rangeAbsorption of the biomolecules < absorption of the water, and dissolving biomolecules in water

♣ “Terahertz excess”: between 1 - 3 THz absorption of the pure biomolecule solids or films generally < absorption of bulk water many situations: the biomolecule+water mixture absorbs > either the biomolecule or a bulk water sample.

Their ascertainments

D.M. Leitner, M. Gruebele, M. Havenith: Solvation dynamics of biomolecules: modeling and terahertz experiments. HFSP Journal Vol. 2, No. 6, December 2008, 314–323

decreasing of the absorption coefficient of the solution at certain frequencies, for proteins in water: ~ 2.5 THz

♣ third substance: biological or hydration water biomolecules perturbs nearby water molecules effect on the properties of water: density, relaxation rates, reorientation rates


15

5. lecture


M. Heyden, E. Brünbenmann, U. Heugen, G. Niehues, D. M. Leitner, M. Havenith: Long-range influence of carbohydrates on the solvation dynamics of water-answers from terahertz absorption measurements and Molecular Modeling Simulations J. Am. Chem. Soc. 130 (17), pp 5773–5779 (2008)B. Born, M. Havenith: erahertz Dance of Proteins and Sugars with Water, J. of. Inf. Mill. and THz Waves, 30, 12, 1245-1254 (2009)

the influence of mono- and disaccharides on the THz spectrum of water

small, isolated saccharide → absorbtion of the THz radiation → at only a few specific frequencies.

saccharides into water → fast oscillations in the water network → in the THz spectrum.



5. lecture


M. Heyden, E. Brünbenmann, U. Heugen, G. Niehues, D. M. Leitner, M. Havenith: Long-range influence of carbohydrates on the solvation dynamics of water-answers from terahertz absorption measurements and Molecular Modeling SimulationsJ. Am. Chem. Soc. 130 (17), pp 5773–5779 (2008)B. Born, M. Havenith: Terahertz Dance of Proteins and Sugars with Water, J. of. Inf. Mill. and THz Waves, 30, 12, 1245-1254 (2009)

the influence of mono- and disaccharides on the THz spectrum of waterfitting the experimental THz absorption coefficients → extension of the dynamical hydration shell around sugars

radii of the shell:glucose: ≈ 4 Ålactose: ≈ 6 Åtrehalose: ≈ 7 Å

amount of water moleculesinfluenced by sugars range:50 water/glucose150 water/lactose190 water/trehalose



5. lecture


M. Heyden, E. Brünbenmann, U. Heugen, G. Niehues, D. M. Leitner, M. Havenith: Long-range influence of carbohydrates on the solvation dynamics of water-answers from terahertz absorptionmeasurements and Molecular Modeling SimulationsJ. Am. Chem. Soc. 130 (17), pp 5773–5779 (2008)B. Born, M. Havenith: erahertz Dance of Proteins and Sugars with Water, J. of. Inf. Mill. and THz Waves, 30, 12, 1245-1254 (2009)

the influence of mono- and disaccharides on the THz spectrum of water

Molecular dynamics simulations: the hydrogen bond dynamics ↔ water molecules around glucose, lactose, trehalose

rearrangement of the hydrogen bonds ↔ water molecules in bulk water to about6 Å away from the solute.



5. lecture


B. Born, K.SJ. Ebbinghaus, M. Gruebele, M. Havenith: The terahertz dance of water with the proteins: the effect of protein flexibility on the dynamical hydration shell of ubiquitin, Faraday Discuss, 141:161-73; discussion 175-207 (2009)S. Ebbinghaus, S.J. Kim, M. Heyden, X. Yu, U. Heugen, M. Guebele, D.M. Leitner, M, Havenith: An extended dynamical hydration shellaround proteins, PNAS,104, 52, 20749–20752 (2007)

hydration study → more complex model system: proteins

highly precise measurements of the THz absorption spectra

spectrum of native ubiquitin wild-type protein in bufferonly buffer



5. lecture


B. Born, K.SJ. Ebbinghaus, M. Gruebele, M. Havenith: The terahertz dance of water with the proteins: the effect of protein flexibility on the dynamical hydration shell of ubiquitin, Faraday Discuss, 141:161-73; discussion 175-207 (2009)S. Ebbinghaus, S.J. Kim, M. Heyden, X. Yu, U. Heugen, M. Guebele, D.M. Leitner, M, Havenith: An extended dynamical hydration shellaround proteins, PNAS,104, 52, 20749–20752 (2007)


>> structural hydration shell, water molecules rigidly attached to protein

globular proteins ubiquitin:

THz absorbance – non-linearto protein concentration

dynamical hydration shell: ≈ 18 Å



5. lecture


S. Ebbinghaus, S.J. Kim, M. Heyden, X. Yu, U. Heugen, M. Guebele, D.M. Leitner, M, Havenith: Protein Sequence- and pH-Dependent Hydration Probed by Terahertz Spectroscopy, J. Am. Chem. Soc., 130 (8), 2374–2375 (2008)


mutants of ubiquitin: the effect of surface properties and flexibility on hydration dynamics

changing the protein to water molar ratios → folded or partially unfolded proteins → distinct response for the THz absorption

synthesized, purified site-specified mutants → at internal side chains →different THz absorbance from natural wild type protein



5. lecture


S. Ebbinghaus, S.J. Kim, M. Heyden, X. Yu, U. Heugen, M. Guebele, D.M. Leitner, M, Havenith: Protein Sequence- and pH-Dependent Hydration Probed by Terahertz Spectroscopy, J. Am. Chem. Soc., 130 (8), 2374–2375 (2008)


mutants of ubiquitin: the effect of surface properties and flexibility on hydration dynamics

changing the protein to water molar ratios → folded or partially unfolded proteins → distinct response for the THz absorption

synthesized, purified site-specified mutants → at internal side chains →different THz absorbance from natural wild type protein



5. lecture

S. Funkner, G. Niehues, D. A. Schmidt, M. Heyden, G. Schwaab, K. M. Callahan, D. J. Tobias, M. Havenith: Watching the Low-Frequency Motions in Aqueous Salt Solutions: TheTerahertz Vibrational Signatures of Hydrated Ions, J. Am. Chem.Soc., 134, 1030−1035 (2012)

relevant question: how the details of ion hydration?

The water and ionic effect

network-coupled dynamics of water in divalent salt solutions

♣ unique high power p-Ge THz laser spectrometer

♣ precise wideband THz Fourier transform (FT) spectroscopy

♣ molecular dynamics simulations

only: atomic anions and cationsavoiding the ambiguities due to coupling between intramolecular ion modes and the water network


5. lecture



♣ unique high power p-Ge THz laser spectrometer

concentration → precise narrow-band THz absorption

strictly linear

from μM to Maveraged between 2.1 and 2.8 THzi.e. 76−93 cm−1

the THz contribution of the solvated ions:αion = αsample - β⋅αwaterαsample : the calculated absorption coefficients using Beer’s law β: the ratio of the number of water molecules at a given concentration in an aqueous solution to that of neat water


5. lecture




each spectrum consists of two absorption peaks which are attributed to either the anion or the cationFor MgCl2 both overlap

liquid cell: 40 μm Kapton spacers, diamond windows nitrogen-purged, temperature-controlled (20 ± 0.1) °Cliquid-He cooled silicon bolometer

concentration of solvated MgCl2: 3 M

spectra of divalent salts


5. lecture




THz ionic absorption bands → fitted to two distinct absorption bandsone corresponding to the anion and one to the cationDots of the same color represent measurements on different concentrations ranging from 0.5 to 4 M


5. lecture



♣ molecular dynamics simulations without getting into details

Symmetry-adapted octahedral normal modes for the Mg2+ ions and their octahedral solvation shells

Vibrational densities of states (VDOS) computed for the projected motion ofMg2+ ions and their first solvation shell along thesymmetry-adapted normal modes.

VDOS computed from Fourier transformed velocity autocorrelation functions in Cartesian space

Mg2+ ions

the oxygens of solvating water molecules

the oxigens of bulk water


5. lecture



♣ molecular dynamics simulations without getting into details

Simulated vibrational density of states (VDOS) computed from Fourier transformed velocity autocorrelation functions of divalent cations and chloride anions


5. lecture


relevant answers: how the details of ion hydration?



♦ the low-frequency (THz) frequency spectrum of a series of salt solutions → linear superposition of concentration weighted neat water and ion contributions

♦ The concentration of the ions → THz absorption: strictly linear

♦ Both anion and cation bands can be assigned independently

♦ coupling between the water molecules in the hydration shell and the ions this lifetime > several vibrational cycles

♦ specific anion and cation resonances: ▬ frequencies ∝ the inverse ion mass and intensities ▬ intensities of these resonances ∝ charge density ▬ these resonances → concerted rattling motions of the anion and cation with its first hydration shell.

be continued


5. lecture


relevant answers: how the details of ion hydration?



♦ very strong coupling of the ions with their first hydration shells

♦ there was not detected any indication of long-ranged effects, which would hint to structure breaking, or structure making, or cooperative effects on water for atomic mono- and divalentsalts

♦ this study underlines the need to include anion and cation contributions for a rigorous analysis of the THz spectrum of solvated salts.

♦ The ions here: ideal, prototypical systems, they lack additional complications, e.g., steric effects and intramolecular vibrations and/or rotational/librational motions


5. lecture

About the experimental methodA. Bergner, U. Heugen, E. Bründermann, G. Schwaab, M. Havenith, D. R. Chamberlin, E. Haller: New p-Ge THz Laser Spectrometer for the Study of Solutions: THz Absorption Spectroscopy of Water, Rev. Sci. Instr. 76, 063110 (2005)

p-Ge laser spectrometer for accurate solvation studies

THz source: semiconductor p-Ge laser, in a closed-cycle helium cryostat, 3-5 Kpulses with up to 1 W peak output power , tuning: 1-4 THzGa:Al detector

window interfaces reflection: varying the layer thichnessabsorption of the sample: increasing exponentiallyreflection unchanged

dual-beam for reference and sample

∙ exp ∙absorbed amount of THz radiation by sample:

Intensity prior to the sample, the frequency dependent absorption coefficient of the samplethe layer thickness d of the sample, the detector offset C.



5. lecture

About the experimental methodA. Bergner, U. Heugen, E. Bründermann, G. Schwaab, M. Havenith, D. R. Chamberlin, E. Haller: New p-Ge THz Laser Spectrometer for the Study of Solutions: THz Absorption Spectroscopy of Water, Rev. Sci. Instr. 76, 063110 (2005)

p-Ge laser spectrometer for accurate solvation studies

THz spectra of hydrated biological samples: 100 400 cmC: constant electronic offset, blocking the leaser beamcontrolled temperature (external water reservoir → sample of temperature: -28 - 55 °C, ±0,05°C) Relative humidity: < 8%; by purging with dry air and nitrogen~ 200 μm thick samples

averaging ~30.000 pulses of 5 μs duration → accuracy of α < 0,1 %, (α ) ± 0,3 cm -1

Precisely measured THz absorption → directly probes the rearrangement of hydrogen bonds

in the water network → THz spectroscopy sensitive detective method:

by biomelcules induced solutes fsat diplole fluctuations of water molecules



5. lecture


E. Bründermann, B. Born, S. Funkner, M. Krüger, M. Havenith: Terahertz spectroscopic techniques for the study of proteins in aqueous solutions Proceedings of SPIE 7215, 72150E1–72150E9 (2009)

THz Fourier transform spectrometer to collect large-bandwidth THz sepctra

Commercial continuous wave FT spectrometer VERTEX 80v, Bruker Co.

For THz source: mercury lampFor beamsplitter: 23 μm mylar folieFor coherent detection: silicon bolometer, cooled by liquid heliumsamples: in a separate compartmentin nitrogen atmosphere


well-known procedure for measurement of the spectrum

I (x) Fourier transformation I(ω)


5. lecture

B. Born, M. Heyden, M. Grossman, I. Saqi, M. Havenith: Protein-water network dynamics during metalloenzyme hydrolysis observed by kinetic THz absorption (KITA) Proceedings of the SPIE, Volume 8585, id. 85850E 7 pp. (2013)

TDTS spectrometer modified for real-time biochemical studieskinetic terahertz absorption spectroscopy KITA


near-infrared femtosecond laser pulses →split into 70:30% for terahertz generation/detectionsample cell of a rapid stopped-flow mixerdetection: electro-optic sampling

for fast mixing of the reagents →stopped-flow sample →following the chemical reactions by kinetic terahertz absorption


5. lecture

B. Born, M. Havenith: Terahertz Dance of Proteins and Sugars with Water, Journal of Infrared, Millimeter, and Terahertz Waves, 30, 12, 1245-1254 (2009)http://www-brs.ub.ruhr-uni-bochum.de/netahtml/HSS/Diss/BornBenjaminPhilipp/diss.pdf


How it works ubiquitin’s folding?

by KITA

denatured ubiquitin

denaturant concentration: 6 M → 0,86 M within milliseconds →♦ refolding of protein♦ triggered reading out of THz electric field

changing of water dielectric properties →changing absorbance →attenuated and shifted THz pulses

GuHCl-denatured ubiquitin + denaturant-free surplus of buffer


5. lecture

B. Born, M. Havenith: Terahertz Dance of Proteins and Sugars with Water, Journal of Infrared, Millimeter, and Terahertz Waves, 30, 12, 1245-1254 (2009)http://www-brs.ub.ruhr-uni-bochum.de/netahtml/HSS/Diss/BornBenjaminPhilipp/diss.pdf


How it works ubiquitin’s folding?

by KITA

Time-dependent electric field of THz pulseskinetic time: mixer is scanned

folding of protein →changing of THz absorbance→changing of THz pulse

refolding kinetics of Ub*at – 20°C in water/ethylene glycol buffer

Documents

Andrea Buzády - PTE-TTK Fizikai Intézet · biomolecular function in vivo ⇔ ⇔ secondary and tertiary structure of bioplimers ⇔ dynamical motions in the relevant timescale at