Enzyme Experiments Using THz Light at the SRS and J-Lab

Hannan Fersi Manchester Interdisciplinary Biocentre The

May 16, 2007 Feasibility Study

Can high power THz radiation be used to search and to probe low frequency protein vibrations that facilitate of hydrogen in systems? Introduction

The physical basis of the catalytic power of remains contentious despite sustained and intensive research efforts

Our knowledge of is predominantly descriptive and gained from traditional protein crystallography and solution studies

The question of whether enzymes have evolved to use quantum tunneling to the best advantage has provoked a heated debate in the Life Sciences Enzyme mechanisms: Where are we at?

- Good appreciation of reaction mechanisms in the terms of bond making/breaking i.e. ‘pushing’ electrons around

- Good appreciation of enzyme structure and static view of mechanism inferred from structural biology approaches And if we are lucky……..

Time-resolved structural methods identify short-lived (in solution) reaction intermediates Less clear – a detailed physical picture of catalysis

• Rate accelerations up to 1018 over reference reaction in absence of an enzyme

• Current physical models (e.g. TST) only account for ~106 What’s missing?

• Protein dynamics that modulate the barrier to the reaction and couple to the reaction coordinate

• Chemical identification of fleeting intermediates e.g. radical species How do enzymes achieve high catalytic rates?

Small-scale promoting vibrations/motions are likely to promote H-transfer and electron transfer by quantum tunneling mechanisms. Probably also true for over-the- barrier mechanisms • Now established that H-transfer by quantum tunneling is widespread in biology

• Controversies remain in the field – how does tunneling barrier compression facilitate the tunnelling process

• Motions in the ES complex occur on the sub picosecond timescale.

• Need to identify motions coupled to the reaction coordinate – needs new experimental methods

• With a few exceptions we are almost entirely reliant on computational methods to identify this type of fast motion Environmentally coupled/vibrationally assisted H-tunnelling model • New theory is emerging to rationalise role of small-scale vibrations in driving enzyme catalysis

• Passive dynamics – no ‘compressive’ motion on the H transfer coordinate. Yields temperature independent KIE

• Active dynamics – a promoting vibration compresses the HC coordinate, narrowing the tunneling barrier to promote tunneling. Yields temperature dependent KIE.

• Active dynamics on sub-picosecond timescale Aromatic Amine Dehydrogenase (AADH) AADH is the most extensively characterised enzyme involved in H-tunnelling (computationally)

In the case of the AADH system the promoting vibration occurs at 165 cm-1

That’ s where THz radiation comes in!! Why we need High Power THZ light…

Spectral investigations are being carried out on a variety of significant biological samples to understand the interaction of far-infrared (FIR) and THz radiation with biological systems on a molecular level, i.e. on the basis of resonant processes with: -Electronic - vibrational - and rotational states of complex biological molecules Why we need High Power THZ light…

• 1 – 10 THz region of the electromagnetic spectrum is particularly difficult to access experimentally due to the low intensity of conventional laboratory based sources

• There is a general interest in developing a new research field linking new powerful sources of THz radiation with key contemporary challenges in protein science

• Specifically, the highly controversial role of fast protein promoting vibrations in biological catalysis Manchester Interdisciplinary Biocentre (MIB) University of Manchester

Dr. Peter Gardner, surface analysis using IR techniques Dr. Nigel Scrutton, mechanisms of redox enzymes * Dr. Mike Sutcliffe, mechanistic and computational enzymology *

Nature 431, 396-397, 2004 * Science (Masgrau et al., in press)*

CLRC Daresbury

Dr. Mark Surman

Dr Gwyn Williams at the Jefferson Laboratory Experimental Approach

SRS Beamline 13.3

Station designed for surface science

Investigations of low frequency adsorbate modes

Martin-Puplet step-scan interferometer P. Gardner recorded spectra down to 100 cm-1

SnCl4 at 120 K SnBr4 at 120 K

101.00 101.00 355 ν4 ν3 355 100.00 100.00 99.00 386 99.00 98.00

133 98.00 97.00 96.00 Inverse

% Reflectance % absorption 97.00 Inverse % reflectance % 95.00 band absorption band 96.00 94.00

93.00 95.00 92.00 ν 417 291 3 94.00 91.00 100 200 300 400 500-1 600 700 200 250 300 350 400 450 500 -1 Wavenumber / cm -1 Wavenumber / cm Good sensitivity to detect single monolayers

100.00

100.00

99.99

99.99

99.98 % Reflectance %

99.98

99.97

99.97 200 250 300 350 400 450 500

Wavenumber / cm -1

Most sensitive far-IR beamline built! Infrared Extraction from the SRS

Collect 60 x 60 mRad Close-in mirror 1 m from tangent point high x-ray power load

The idea!!

With minor modifications the station 13.3 will be converted into a dedicated THz test facility to see if it is possible to detect the promoting vibrations Aim on the study

• Reconfigure THz test facility to incorporate the second optical bench optimised for 100 – 300 cm-1 region and commission this system

• Obtain a high resolution spectrum from AADH in the 10 – 300 cm-1 region to characterise modes in different forms of AADH to identify the modes important in the H-tunnelling reaction

• Pump probe these modes to identify which of these motions act as rate promoting vibrations for the H-tunnelling reaction

• Life-time analysis of promoting mode(s) in active enzyme- substrate complex and inactive enzyme-substrate analogue complex as further evidence for a role for vibrations in the H- tunnelling reaction Sample preparation for solids

The compounds were mixed with PET or PTFE powder and grind to reduce particle size

The IR pellets were made up into a 13 mm diameter, self supporting pressed disc, with a 0.5 mm to 1 mm thickness using ~2 ton pressure

Background spectrum of pure PET or PTFE

All the spectra were collected in the range of 1000 to 25 cm-1 (0.5 cm-1 resolution) Sample preparation for solutions

Bovine serum albumin (BSA) is our representative biomolecule

BSA is a monomeric protein of molecular weight 66.4 kDa and shares 76% sequence homology with human serum albumin Its PDB code is 1AO6 (Huang et al. 2004)

Proteins solutions will be made by dissolving X mg of lyophilized powders of proteins X into Tris buffer at pH 7.0.

Homogeneity of the solutions is critical for THz measurements so the solutions will be sonicated for 1 h to fully dissolve aggregates.

The solutions will be injected into the FTIR cell Sample preparation for solutions

One cell for different pathlengths:

• Rotation of the main body alters the pathlength of the cell over a range of 0 mm to 6 mm with scale division of 5µm

• The windows do not rotate in relation to the body movement and hence maintain parallelism throughout the pathlength range

• Easily increase pathlength to observe minor bands Uric acid THz spectra The first test spectra using the THz test facility on the SRS for THz spectroscopy were recorded on uric acid in order to try to reproduce data obtained in the THz Bridge program.

Uric acid

Uric acid spectrum obtained using THz time domain spectroscopy from solid pellets Taday et al Spectrum of uric acid and allantoin at 0.5 cm-1 spectral resolution

277 cm-1 163 cm-1 297 cm-1

144 cm-1

133 cm-1

48 cm-1 -1 215 cm 80 cm-1 47.9 cm-1

79.6 cm-1 Absobance

40.2 cm-1

Gardner et al

1.0 1.5 2.0 2.5 3.0 Frequency / THz

Taday et al Bovine Serum Albumin (BSA) THz spectra

Jing Xu, Kevin W. Plaxco and S. James Allen 2006 15: 1175-1181 Protein Sci.

2.0

BSA/PTFE 20/80 %(w/w) 1.5 Nic0218new.spa

BSA/PTFE 50/50 %(w/w) 1.0 Nic0253new.spa Absorption 0.5

0.0

1.0 1.5 2.0 2.5 3.0 Frequency / THz Summary

• Station 13.3 has been converted into a working THz spectroscopy test facility

• We obtained THz spectra of uric acid, allantoin,L-glutamic acid, L and D glucose, sucrose, tryptamine and BSA.

•First tests suggest that it is working as well if not better than expected

• First experiments with small molecules and BSA at high resolution are consistent with previous work and very encouraging.

• Still early days Goal for our first visit at J-Lab

• Reproduce THz spectra of simple solids and BSA

• Do preliminary studies of small molecules and proteins in solutions

• The signal-to-noise ratio in the spectrum is likely to be poor in this region so optimisation of pathlength/solvent/ concentration etc.

• Does THz light at J-lab damage our samples? What are our limitations? Should we use a flow cell? Future work at J-lab

• THz facility at the Jefferson Laboratory is the most powerful since it can deliver both high peak power and high repetition rate

• Obtain THz spectra of more solid proteins and proteins in solution

• Identify band at 165 cm-1 in AADH system

• Carry out pumping experiments at J-Lab and attempt to influence reaction rates Acknowledgements

• Manchester Interdisciplinary Biocentre, University of Manchester

Dr. Peter Gardner Dr. Nigel Scrutton Dr. Mike Sutcliffe

• CLRC Daresbury

Dr. Mark Surman Dr. Terry Lee Technical Staff

• Jefferson Laboratory, FEL Dr Gwyn Williams

EPSRC Physics at the Life Science Interface