Research Project
10317725
Summary The Sosnick lab has studied the dynamics and function of soluble proteins using an integrated experimental and computational approach for the past 25 years. This proposal expands our focus to include membrane proteins with an emphasis on applying hydrogen-deuterium exchange (HDX) to study the relationship between dynamics and function. In Aim 1, we will address a central challenge in protein biophysics ? apply and rigorously test our ability to accurately simulate the free energy surface including the generation of the Boltzmann ensemble of all major species. The lab has made considerable progress in this area, developing Upside, a molecular dynamics algorithm that can cooperatively fold proteins with an accuracy comparable to all-atom methods yet is 103-104 fold faster. The algorithm will be advanced, validated, and applied across the three specific aims. Testing will be done largely using HDX/NMR as this powerful combination of methods can determine the free energies of individual H-bonds throughout a protein and quantitatively describe the free energy surface with high precision. Test systems include designed and naturally occurring proteins. In Aims 2 and 3, we will continue our studies of the folding and dynamics of KcsA and other ion channels in liposomes, developing methods to measure HDX on membrane proteins during folding and activation by voltage, pH and tension to explore the relationship between their dynamics and function. Preliminary results on the folding of the KcsA find that the initial folding event is a rapid association into a protein-dense phase, while the rate-limiting step in forming the native tetramer is a unimolecular event. We will continue to investigate the protein-dense phase and the overall folding kinetics, testing our prediction that the rate-limiting step involves the formation of a solvent channel followed by insertion of the four selectivity filters and pore helices. To identify when specific H-bonds form during KcsA folding, we will use HDX pulse-labeling with mass spectrometry and thus shed light on this final pathway to the tetramer. Finally, it is often the case that structural biologists can only obtain the structure of a membrane protein in either its resting or activated state, and even if both structures are known, the dynamics and pathways between the states remain to be determined. We propose to develop robust protocols to obtain HDX patterns of both states for KcsA, KvAP, and the mechano-sensing channel MscS using HDX/mass spectrometry to identify the structural dynamics associated with the proteins? functional processes. In doing so, we will further expand the repertoire of applications in which the HDX method, so rich in information content, can be applied. 2
