Session H7: Physics of Proteins II: Dynamics and Functions

Energy Landscapes Encoding Function in Enzymes Investigated Over Broad Time Scales

The operating hypothesis of much of our current work is that atomic motion, over broad time scales (femtoseconds to milliseconds, the latter being the time scale of most enzyme catalyzed reactions), contributes to enzymic catalysis in proteins. It is clear from our work that specific types of motions are important in binding of ligands to proteins and transition state formation in enzymatic catalysis. Since new experimental and theoretical approaches are needed to understand the dynamical nature of proteins broadly and enzymatic catalysis specifically, we have employed time-resolved ``pump-probe'' spectroscopic techniques because of the sensitivity of these type of approaches to all relevant time scales. And we have also developed and applied new theoretical methods. The talk will focus on how lactate dehydrogenase brings about catalysis based on current experimental and theoretical studies.   

NMR investigations of molecular dynamics

NMR spectroscopy is a powerful experimental approach for characterizing protein conformational dynamics on multiple time scales. The insights obtained from NMR studies are complemented and by molecular dynamics (MD) simulations, which provide full atomistic details of protein dynamics. Homologous mesophilic (\textit{E. coli}) and thermophilic (\textit{T. thermophilus}) ribonuclease H (RNase H) enzymes serve to illustrate how changes in protein sequence and structure that affect conformational dynamic processes can be monitored and characterized by joint analysis of NMR spectroscopy and MD simulations. A Gly residue inserted within a putative hinge between helices B and C is conserved among thermophilic RNases H, but absent in mesophilic RNases H. Experimental spin relaxation measurements show that the dynamic properties of \textit{T. thermophilus} RNase H are recapitulated in \textit{E. coli} RNase H by insertion of a Gly residue between helices B and C. Additional specific intramolecular interactions that modulate backbone and sidechain dynamical properties of the Gly-rich loop and of the conserved Trp residue flanking the Gly insertion site have been identified using MD simulations and subsequently confirmed by NMR spin relaxation measurements. These results emphasize the importance of hydrogen bonds and local steric interactions in restricting conformational fluctuations, and the absence of such interactions in allowing conformational adaptation to substrate binding.   

Kinetic vs. Thermodynamic Control of Bacteriorhodopsin Pumping

Bacteriorhodopsin is a transmembrane proton pump that converts light energy to a transmembrane electrochemical gradient. Retinal, bound in the center of the protein, absorbs light and isomerizes from the all-trans to 13-cis configuration. A series of conformational changes and proton transfers then restores the structure to the all-trans ground state while pumping one proton from the high pH cell interior to the low pH exterior, saving energy in an electrochemical gradient. Poorly understood gating elements control key steps where incorrect proton transfer would return the protein to the ground state without pumping. The gate's barrier height determines how much the pump leaks. Analysis of high-resolution structures trapped in different intermediates has produced ideas for how bacteriorhodopsin ensures pumping. There are two contrasting strategies, one primarily thermodynamic and the other relying on kinetic control to ensure that protons are moved uphill. With thermodynamic control, residue protonation states always remain in quasi-equilibrium. Relatively slow conformational changes shift the energy landscape modifying site pKas. Residues then change ionization remaining in equilibrium in each metastable intermediate. The sequence of intermediates imparts the directionality to the transfers. Alternatively, the direction of transfer is determined by the accessibility of low energy pathways so is thus is under kinetic control. We will discuss which steps in the bacteriorhodopsin photocycle are under thermodynamic or under kinetic control. The role of three specific conformational changes (retinal isomerization, Arg82 reorientation and Glu194 and 204 separations) on the degree of proton transfer will be described. Supported by NFS MCB 1022208.   

Beller Lectureship Talk: Ultrafast Excitation Energy Transfer and the Mechanism of Non-Photochemical Quenching in Plant Photosynthesis

The success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. LHCII, the peripheral light-harvesting complex of Photosystem II, plays a major role. At the same time, the same light-harvesting system can be `switched' into a quenching state, which effectively protects the reaction center of Photosystem II from over-excitation and photodamage. In this talk I will demonstrate how LHCII collects and transfers excitation energy. Using single molecule spectroscopy we have discovered how LHCII can switch between this light-harvesting state, a quenched state and a red-shifted state. We show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under `NPQ-like' conditions. It is argued that this is the mechanism of non-photochemical quenching in plants.   

Protein Dynamics, Ligand Binding, and Biological Function

Dynamics is essential for protein function. To demonstrate this point, this talk presents three studies. (1) For a ligand-gated ion channel, ligand binding leads to channel activation by modulating the dynamics of the channel protein. A common theme that emerges from different families of ligand-gated ion channels is that agonist binding closes the ligand-bidning domain (LBD), leading to pore opening in the transmembrane domain (TMD); in contrast, antagonist binding opens the LBD, leading to pore closing in the TMD [1]. (2) When the structure [2] and gating dynamics [3] of the influenza M2 proton channel are accounted for, the calculated rate of ion transport is in quantitative agreement with experimental data [4]. (3) In enzymes, gating dynamics afford substrate selectivity [5]. \\[4pt] [1] M. Yi, H. Tjong, and H.-X. Zhou (2008). Spontaneous conformational change and toxin binding in $\alpha $7 nicotinic acetylcholine receptor: insight into channel activation and inhibition. Proc. Natl. Acad. Sci. 105, 8280-8285. \\[0pt] [2] M. Sharma, M. Yi, H. Dong, H. Qin, E. Peterson, D. D. Busath, H.-X. Zhou, and T. A. Cross (2010). Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer. Science 330, 509-512. \\[0pt] [3] M. Yi, T. A. Cross, and H.-X. Zhou (2009). Conformational heterogeneity of the M2 proton channel and a structural model for channel activation. Proc. Natl. Acad. Sci. USA 106, 13311-13316. \\[0pt] [4] H.-X. Zhou (2010). Diffusion-influenced transport of ions across a transmembrane channel with an internal binding site. J. Phys. Chem. Lett. 1, 1973-1976. \\[0pt] [5] H.-X. Zhou, S. T. Wlodek, and J. A. McCammon (1998). Conformation gating as a mechanism for enzyme specificity. Proc. Natl. Acad. Sci. USA 95, 9280-9283.