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Characterization of membrane protein dynamics by hydrogen/deuterium exchange and time-resolved infrared spectroscopy, assisted by maximum entropy and Bayesian methods of analysis

Periodic Report Summary 1 - NAGOYA2BCN (Characterization of membrane protein dynamics by hydrogen/deuterium exchange and time-resolved infrared spectroscopy...)

Objectives

The main objective of this project is to obtain insights on how and to which level protein dynamics may alter the ground state structure in membrane proteins, as well as to characterise the transient protein conformations involved in protein function.

We propose the use of hydrogen/deuterium exchange (HDX) monitored by infrared (IR) spectroscopy to characterise fluctuation in membrane proteins under native conditions. This requires some technical improvements over previous implementations of HDX using IR spectroscopy, as well as improvements/developments in the way to analyse the HDX data in order to obtain detailed information on the dynamics. The membrane proteins considered for such characterisation are bacteriorhodopsin (bR), the melibiose permease (melB), and a GPCR chimera.

To study the dynamics of protein involved in protein function, we propose to study the proton-pump mechanism of bacteriorhodopsin (bR). In spite of many efforts, it has not been yet possible to reliably resolve the conformation of the transiently populated intermediates nor the dynamics of their interconversion. The application/development of maximum entropy and Bayesian methods is expected to provide new light in these aspects.

Work performed and main results

Hydrogen/deuterium exchange (HDX).
Useful HDX data requires changing the buffer from a H2O to a D2O medium as fast as possible, without back-contamination in the long term. As the time window covered in the HDX experiments increases, so do the time-scale (and energy-scale) of protein fluctuations that can be monitored and characterised. We have struggled to design an experimental setup for IR spectroscopy able to improve the time-window of HDX experiments. Using attenuated total reflection (ATR) cell, we developed a simple approach able to change the protein from a H2O medium to a 95 % D2O medium in few seconds. This represents an improvement in resolution of more than one order of magnitude to respect previously described HDX setups. Moreover, an almost null level of back-contamination maintained up to >12 h. We have used this set up to perform HDX experiments for bacteriorhodopsin at room-temperature at several pH values.

In a recent collaboration with Prof. Kandori in Nagoya (Japan), Bayesian inference with Markov chain Monte Carlo sampling was used to globally analyse the HDX of the cone receptor rhodopsin as a function of the pH and the temperature (Lorenz-Fonfria et al., submitted). The dynamics and thermodynamics of a fluctuation that affects a residue in steric contact with the light-sensing retinal molecule was fully characterised, and found to correlate with previous estimates of the frequency and activation energy for the dark-activation of rhodopsin. We hypothesise that this protein fluctuation may be involved in the false perception of photons in total darkness.

Analysis of the bacteriorhodopsin photocycle.
Attempts have been performed to analyse time-resolved IR data of the bR photocycle using the inverse Laplace transform (iLT) with the maximum entropy method (MaxEnt). But the interpretation of the results are pending until we also develop appropriate tools for the identification of which features of the obtained 2D-lifetime distribution are reliable and which ones may be induced by noise.

Improvement/development of methods.
We have worked in the improvement/development of some tools and software to facilitate the analysis of the HDX data and the bR photocycle. These improvements have been included in a visual program running in Matlab. We have also done work and progress in the theoretical characterisation of Fourier deconvolution (Lorenz-Fonfria & Padros, 2009 Appl Spectrosc). This mathematical process allows narrowing IR bands, providing more resolved spectra, and thus more 'resolved' structural information.