Vibrational Coupling as a Probe of Biomolecular Structure and Dynamic
Final Report Abstract
Structure and function of complex biomolecules and biologically important compounds are explored by utilizing the coupling between molecular vibrations as a highly specific probe of the local molecular environment. We use emerging multi-dimensional Infrared spectroscopies to measure the time-evolution of molecular structure and vibrational coupling at equilibrium and during chemical reactions. The data is displayed in two-dimensional frequency maps, correlating coupled vibrational modes, similar to how 2D-NMR correlates coupled nuclear spins. Spectrally isolated localized vibrational modes are being used to define a molecular reference frame, with respect to which the orientation of other molecular vibrational modes are determined. By comparing experimental 2D-IR spectra to high order vibrational calculations a three-dimensional molecular picture of the underlying dynamic is reconstructed. Emphasis is placed on the dynamics of peptide models such as the cyclic amide 2-pyrrolidinone, and porteins such as tumor suppressor protein p53 and its molecular chaperon HSP90. We reported about the problems with the commercial laser system which led to low signals and therefore we had to stop our experiments on the proteins p52 and HSP90 and move to high signal model peptides like 2-pyrrolidinone. The mid-IR spectrum of the hydrogen bonding lactam 2-pyrrolidinone in CCl4 was studied using FT-IR spectroscopy accompanied by a quantum chemical anharmonic normal mode analysis combined with a Monte Carlo approach based on semi empirical harmonic frequencies. We characterize and assign the spectroscopic features in the range from 1500 cm−1 to 3600 cm−1 covering the respective amide bands related to hydrogen bonding CO and NH groups as well as the backbone CH vibrational band. Comparing theory and experiment we are able to assign all mid-IR features to a variety of distinct structures of 2-pyrrolidinone, ranging from monomers, singly and doubly hydrogen bonded dimers to longer hydrogen-bonded chains. These chains account for the most prominent features in the IR spectrum. Furthermore, two peaks in the CH band have been identified as highly localized axial and equatorial C-H stretch vibrations qualified for probes of conformational dynamics. The measurements on methylbenzoate revealed only very low correlation of the carbonyl band to the backone C-H/D bands. For most chemical and biological applications the information about energy flow, population relaxation and transfer is more important than the knowledge of coherences in a molecule. Therefore, we also performed 2D echo experiments that are sensitive to population transfer, the T-scan experiment. To our knowledge we are the first group to perform such experiments, were we prepare a specific population state, by selecting an appropriate tau delay, and scan the time behaviour of that specific population state. A variation of that technique, called accordion, is very popular and successful in NMR spectroscopy. The molecular chaperone Hsp90 is responsible for the stabilization and activation of a large number of key regulatory proteins such as the tumor suppressor factor p53, steroid hormone receptors, kinases and a variety of unrelated proteins which have been identified during the last decade. To directly measure the predicted folding/unfolding equilibrium process of HSP90 we constructed seven Cystein point mutants of the N-terminal Domain of Hsp90 (Aminoacids 1-210). The Cysteins have been placed in the region where the strongest structural fluctuations are predicted. Due to break down of the commercial laser systems we were not able to reproduce our results. The superstable quadruple mutant of the p53 core domain (M133L/V203A/N239Y/N268D) was generated and expressed in E. coli yielding good amounts of protein. Due to break down of the commercial laser systems we were not able to generate 2D IR spectra.
Publications
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Hydrogen bonding characteristics of 2-Pyrrolidinone: A joint experimental and theoretical study, Phys. Chem. Chem. Phys., (2012)
Kiran Sankar Maiti, Andriy Samsonyuk, Christoph Scheurer and Tobias Steinel
