NAGOYA2BCN

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

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 Obiettivo del progetto (Objective)

'Protein dynamics is essentially linked to protein function, aggregation, and folding. Time-resolved spectroscopy is specially suited to reveal these dynamics, counterbalancing the static pure-structural information provided by X-ray crystallographic structures. In equilibrium, protein conformation fluctuates, eventually visiting all possible higher-energy states in equilibrium with the ground state conformation. For soluble proteins the dynamics of such fluctuations have been successfully detected and characterized by hydrogen/deuterium exchange (HDX) combined with NMR spectroscopy. However, experimental limitations of NMR spectroscopy have precluded a similar level of understanding of the dynamics of membrane proteins. Proteins dynamics manifest themselves also when a fast perturbation is applied, such as when a laser pulse is applied to proteins with photocycles. Both in HDX and in perturbation experiments the time-resolved response of the system is a multi-exponential relaxation process. However, the relevant information to characterize and understand the protein dynamics is not the relaxation process itself, but the number, value, and nature (discrete/distributed) of the rate constants of the relaxation process. In this project our first goal is the improvement, development and application of maximum entropy and Bayesian methods to analyze the multi-exponential data arising in the HDX of membrane proteins, and in the photocycles of membrane proteins. Simultaneously, we will implement new experimental approaches of HDX to obtain dynamics information of membrane proteins by infrared spectroscopy, and apply them to at least three membrane protein systems: the melibiose transporter, bacteriorhodopsin, and a G-protein coupled receptor chimera. The combined success of both interdisciplinary goals should provide unique and fundamental information for a better understanding of membrane protein dynamics.'

Introduzione (Teaser)

The dynamics of conformation fluctuations for soluble proteins have been successfully detected and characterised. Experimental limitations of NMR spectroscopy, however, do not afford an equal level of understanding of the dynamics of membrane proteins.

Descrizione progetto (Article)

Protein dynamics is basically linked to protein function, aggregation and folding, and time-resolved spectroscopy is especially suited to reveal these dynamics. Protein dynamics also manifest themselves when a fast perturbation is applied. An example of this is when a laser pulse is applied to proteins with photocycles.

Both in hydrogen/deuterium exchange (HDX) and in perturbation experiments the time-resolved response of the system is a multi-exponential relaxation process. The information to be characterised is the number, value and nature of the rate constants of the relaxation process, not the process itself.

The NAGOYA2BCN project aimed to improve, develop and apply maximum entropy and Bayesian methods to analyse the multi-exponential data arising in the HDX of membrane proteins, and in the photocycles of membrane proteins. At the same time, it set out to use new experimental HDX approaches to obtain dynamics information of membrane proteins by infrared (IR) spectroscopy, and then apply them to at least three membrane protein systems: the melibiose transporter, bacteriorhodopsin, and a G-protein coupled receptor chimera.

Results being reported reveal that the time-scale of protein fluctuations that can be monitored and characterised increases relevant to a longer time window covered in the HDX experiments. Although some obstacles were encountered in designing an experimental set-up for IR spectroscopy to improve the time-window of HDX experiments, the research team developed a simple approach changing the protein from a H2O medium to a 95% D2O medium in a few seconds.

Achievements have also been noted during collaboration with Prof. Kandori in Nagoya, Japan, in Bayesian inference with Markov Chain Monte Carlo sampling.

In other work, headway has been made in improving/developing various tools and software that facilitate the analysis of the HDX data and the bR photocycle. These improvements have been included in a visual program running in Matlab. The successes of the NAGOYA2BCN project offer unique and essential information to better grasp membrane protein dynamics.