Enzyme Experiments Using THz Light at the SRS and J-Lab
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1 Enzyme Experiments Using THz Light at the SRS and J-Lab Hannan Fersi Manchester Interdisciplinary Biocentre The University of Manchester May 16, 2007
2 Feasibility Study Can high power THz radiation be used to search and to probe low frequency protein vibrations that facilitate quantum tunnelling of hydrogen in enzyme systems?
3 Introduction The physical basis of the catalytic power of enzymes remains contentious despite sustained and intensive research efforts Our knowledge of enzyme catalysis 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
4 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
5 And if we are lucky.. Time-resolved structural methods identify short-lived (in solution) reaction intermediates
6 Less clear a detailed physical picture of catalysis Rate accelerations up to over reference reaction in absence of an enzyme Current physical models (e.g. TST) only account for ~10 6
7 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
8 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-thebarrier mechanisms
9 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
10 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
11 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!!
12 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
13 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
14 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, , 2004 * Science (Masgrau et al., in press)* CLRC Daresbury Dr. Mark Surman Dr Gwyn Williams at the Jefferson Laboratory
15 Experimental Approach SRS Beamline 13.3 Station designed for surface science Investigations of low frequency adsorbate modes Martin-Puplet step-scan interferometer
16 P. Gardner recorded spectra down to 100 cm -1 SnCl 4 at 120 K SnBr 4 at 120 K % reflectance ν 4 ν 3 Inverse absorption band 355 % Reflectance Inverse absorption band Wavenumber / cm ν Wavenumber / cm -1
17 Good sensitivity to detect single monolayers % Reflectance Wavenumber / cm -1 Most sensitive far-ir beamline built!
18 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
19 Aim on the study Reconfigure THz test facility to incorporate the second optical bench optimised for cm -1 region and commission this system Obtain a high resolution spectrum from AADH in the 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 enzymesubstrate complex and inactive enzyme-substrate analogue complex as further evidence for a role for vibrations in the H- tunnelling reaction
20 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)
21 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
22 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
23 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
24 Spectrum of uric acid and allantoin at 0.5 cm -1 spectral resolution 297 cm cm cm cm cm cm cm -1
25 47.9 cm -1 Absobance 79.6 cm cm -1 Gardner et al Frequency / THz Taday et al
26 Bovine Serum Albumin (BSA) THz spectra Jing Xu, Kevin W. Plaxco and S. James Allen : Protein Sci. 2.0 Absorption BSA/PTFE 20/80 %(w/w) Nic0218new.spa BSA/PTFE 50/50 %(w/w) Nic0253new.spa Frequency / THz
27 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
28 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?
29 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
30 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
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