Session H17: Proteins: Structure and Function

Linking enzyme conformational dynamics to catalytic function with single-molecule FRET

Many enzymes endure sizable conformational remodeling on a timescale comparable to their catalytic cycle. These conformational dynamics may be critical to the enzymes' catalytic function. In adenylate kinase (AK) from \textit{E. coli}, this involves a large-amplitude rearrangement of the enzyme's lid domain. We use high-resolution single-molecule FRET developed in our laboratory to measure AK's domain movements on its catalytic timescale. We utilize maximum entropy-based methods to remove photon-counting noise from raw data, so that the enzyme's entire conformational distribution can be quantitatively recovered without a presumed model. Multiple sequence alignment suggests regularities between the conserved residues and their structural-functional roles. Armed with precise single-molecule FRET dynamics measurements and comprehensive bulk kinetic studies of the mechanism, we were able to quantitatively correlate AK's stochastic lid dynamics with its deterministic catalytic rates. Implications on the structure-function conservation and protein engineering will be discussed.   

Concentration-dependent Cu(II) binding to prion protein

The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.   

Solvable model of mechanical unfolding of proteins

We present exact analytical results describing single-molecule experiments on mechanical unfolding of proteins within a realistic model [1]. The corresponding relation between the extension at a given temperature of the macromolecule and the applied force is derived [2]. The configuration partition function is calculated exactly for a distance constraint protein model that describes the beta-hairpin to coil transition. The resulting extension-force curve is derived, and the results agree with the data from the single-molecule pulling experiments. \newline [1] O.K.Vorov, A.Y.Istomin, D.R.Livesay, D.J.Jacobs, subm. to Phys.Rev.Lett., 2007. [2] O.K.Vorov, D.R.Livesay, D.J.Jacobs, to be subm. to Science, 2007, in preparation.   

Dynamical Transition in polypeptides

Two of the possible causes for the so called dynamical transition (the rapid increase in flexibility for biomolecules at $\sim $ 200 K) are: thermally activated side chain diffusive motions with hydration dependent activation energies; or a glass transition in the biological water directly adjacent to the biomolecule. If the transition is strictly due to side chain activation, it should not depend on protein structure. Previously we demonstrated that the dynamical transition remains after tertiary structure was removed using THz time domain dielectric spectroscopy (0.2 -2.0 THz, 0.5-5ps). Here measurements on polyalanine as a function of chain length show that the dynamical transition does not occur for peptide length shorter than 5. However, the transition is observed for 5 mer and higher. Structural and simulation studies indicate that the 5 mer transiently occupies structured forms [1,2]. These results suggest that A) the dynamical transition is not due to thermally activated side chain motion and B) secondary structure is necessary for the dynamical transition. Secondary structure possibly induces sufficient ordering in the adjacent water to result in a fragile to strong glass transition resulting in increased protein flexibility [3]. [1] KAH Wildman et al. Solid State Nucl. Magn. Reson. 24 (2003) 94-109. [2] Yuguang Mu,et al. Proteins 58, (2005) 45-52. [3] S.H. Chen et al. PNAS (2006) 9012--9016.   

Dynamically stable beta-sheets in Cu-initiated misfolding of $\alpha$-synuclein

The human protein $\alpha$-synuclein has been implicated as a central constituent in multiple neurodegenerative diseases. In Parkinson disease it is even thought to be the causative link. $\alpha$-synuclein can be stimulated to aggregate into deleterious fibrillar structures by mutation, metal binding, and agitation. In particular, Cu$^{2+}$ has been found in high concentrations in neural tissues of Parkinson sufferers. We propose a scenario involving the metal ion Cu$^{2+}$ as the misfolding $\beta$-sheet initiator of fibrillogenesis. A model fragment of the metal-bound protein was investigated using DFT to obtain conformational details of the energetically favorable geometries. Feasible $\beta$-sheet structures incorporating the DFT geometries were explored using heuristic $\beta$-sheet guidelines and inverse kinematics. The resulting structures were tested for dynamic stability by simulating the fully solvated protein by classical MD constrained by the DFT geometries. Our results indicate that dynamically stable structures exist and that the metal binding is directly responsible for initiating misfolding.   

Dynamics of Lysozyme in Trehalose solutions

Anhydrobiosis in Tardigrades and Nematodes has been a topic of constant interest and intrigue in the scientific community. An increase in the concentration of Trehalose has been attributed to the ability of some organisms to survive extreme conditions of temperature, pressure and pH. Although there exist many experimental studies attributing this effect to Trehalose, the molecular details governing the interaction between Trehalose and proteins remains unclear. We have conducted a 20ns study of Lysozyme in varying concentrations of Trehalose in water. Strong and weak hydrogen bonds and hydrophobic interactions between water, Trehalose and protein seem to dictate the interactions in the system. We have observed a hydrogen bonded network of Trehalose around the protein entrapping a layer of water between itself and protein. Lysozyme remains in a near-native conformation throughout the simulation giving hints on the ability of Trehalose in preserving the structure of protiens.   

Spectral and Hydration Dependence of Protein Dynamical Transition

The protein dynamical transition, a rapid increase in flexibility at $\sim $ 200K, is hydration dependent suggesting that the transition may in fact be due to a transition in the surrounding water. Previously we have shown that the terahertz dielectric response is sensitive to the dynamical transition using terahertz time domain spectroscopy [1]. The broadband technique allows the determination of what motions are affected by the transition, that is whether long time scale motions such as side chain rotations, or faster vibrational motions. Here we examine both the frequency and hydration dependence of the protein dynamical transition for hydrated myoglobin powder for the 0.2 -- 2.0 THz and 80-295 K ranges. The transition is observed in both the real and imaginary parts of the dielectric response. Our earlier measurements of solutions did not show a transition in the real part of the permittivity, likely due to bulk solvent dominating the index. There is a strong frequency dependence with hydration. While a slight transition is observed at frequencies higher than 1 THz which is nearly hydration independent, for frequencies below 1 THz the strength of the transition rapidly increases with hydration. [1] A. G. Markelz, J. R. Knab, Jing Yin Chen, Yunfen He, Chem. Phys. Lett. \textbf{442}, 413 (2007).   

Vibrational Dynamics of Heme Model Compounds

Synchrotron- and laser-based measurements supported by DFT calculations identify vibrational modes of the iron atom in Fe(P)(Cl) and Fe(P)(Br). These compounds are large enough to capture many essential aspects of heme geometry and vibrations. On the other hand, porphine models are small enough to simplify the vibrational spectrum and enable accurate analysis using DFT methods. Nuclear resonance vibrational spectroscopy (NRVS) and femtosecond coherence spectroscopy (FCS) provide a rare opportunity to identify both doming and Fe-halide stretching components of the reaction coordinate with confidence. Correlation analysis between 4-coordinate and 5-coordinate compounds suggests significant mixing between Fe-ligand and heme modes. Comparison with the larger model Fe(III)(OEP)(Cl) reveals the effect of peripheral group substitutions.   

Ligand-Modulated Free Energy Landscapes of Glucose/Galactose Binding Protein

Glucose/galactose binding protein (GBP) functions as part of a larger system of proteins for molecular recognition and signaling in enteric bacteria. Here we report on the thermodynamics of conformational equilibrium distributions of GBP from both time-resolved fluorescence experiments and computational umbrella sampling molecular dynamics analyzed by the weighted histogram analysis method (WHAM). Three conformations appear at zero glucose concentration and systematically transition to three conformations at high glucose concentration. Fluorescence anisotropy correlations, fluorescent lifetimes, thermodynamics, computational structure minimization and molecular dynamics, and previous work were used to identify the three components as open, closed, and twisted conformations of the protein. The existence of three states at all glucose concentrations indicates that the protein continuously fluctuates about its conformational state space via thermodynamically driven state transitions, and the glucose biases the populations by reorganizing the free energy profile. These results and their implications are discussed in terms specific and non-specific interactions GBP has with cytoplasmic membrane proteins.   

Ligand Binding Kinetics in Myoglobin and Solvent Relaxation at High Pressure

Pressure is increasingly used as a variable to examine protein structure-function relationships, since it is crucial for chemical equilibria, reaction rates, and protein conformational states. We investigate pressure effects for the prototype reaction of ligand binding to myoglobin over a wide dynamic range in time and temperature. The distribution of rebinding rates is evaluated from kinetic absorption measurements of CO and O$_{2}$ binding to (horse) myoglobin at variable pressure (0.1 - 190 MPa) and temperature (180 - 300 K) in aqueous and 75 {\%} glycerol/buffer solutions. The data demonstrate that pressure significantly affects the amplitudes (not just the rates) of the component processes. The amplitude of the geminate process increases with pressure corresponding to a smaller escape fraction of ligands into the solvent and a smaller inner barrier. Solvent relaxation rates at variable pressure are determince independently from specific heat spectroscopy. We discuss the role of solvent dynamics, hydration shell, and internal protein cavities in the binding reaction.   

Electronic Structure and the Magnetic Hyperfine Interactions in Heme Unit of Metmyoglobin

The $^{14}$N and $^{57m}$Fe hyperfine interactions in the heme unit of metmyoglobin are available experimentally by electron-nuclear double resonance (ENDOR) and Mossbauer spectroscopic techniques. We have carried out electronic structure investigations on the heme system including the H$_{2}$O and proximal imidazole ligands by the first-principles Hartree-Fock procedure and studied the magnetic hyperfine and nuclear quadrupole coupling constants for the $^{57m}$Fe nucleus and all the six $^{14}$N nuclei on the four pyrrole and imidazole ligands as well as the $^{17}$O nucleus on the H$_{2}$O ligand. Comparison will be made with available experimental data [1, 2] and earlier theoretical investigations [3] by the approximate self-consistent charge Extended Huckel procedure. Results will also be presented for the optical frequencies and intensities from transitions between ligand-like and iron d-like states and the Fe-N$_{\varepsilon }$ vibrational frequency [1] G. Lang, Q. Rev. Biophys. \underline {3}, 1 (1970) [2] C.P. Scholes, R.A. Isaacson and G Feher, Biochim. Biophys. Acta \underline {263},448(1972) [3] S.K. Mun, Jane C. Chang and T.P. Das J. Am. Chem. Soc. \underline {101}, 5562(1979)   

Hartree-Fock Investigation of Electronic Structure and Associated Properties of Heme Unit in Deoxyhemoglobin

Using the Hartree-Fock-Roothaan procedure and the most recent version of the Gaussian set of programs we have studied the electronic structure of the heme unit including the imidazole ligand of iron from the proximal histidine using x-ray data for the positions of all the atoms except the hydrogen. The positions of the latter have been obtained through energy optimization. The results obtained from the calculated electronic structure for the magnetic and electronic quadrupole hyperfine interactions of $^{57m}$Fe and $^{14}$N nuclei will be discussed. Comparison will be made with available experimental data and earlier theoretical investigations [1]. Results will also be presented for the proximal Fe-N$_{\varepsilon }$ vibrational frequency and the frequencies and intensities of optical transitions between ligand like states and d-like states of Fe [1]. K Ramani Lata PhD Thesis SUNY Albany (1993) (Unpublished)   

The Electronic Investigation of Singlet and Triplet States of Oxyhemoglobin by Hartree-Fock Procedure and Associated Hyperfine Interaction.

The observation of paramagnetic susceptibility [1] in Oxy-Hb from measurements over a broad temperature range has stimulated interest in the occurrence of a low-lying excited triplet state close to the ground singlet state of Oxy-Hb. An earlier theoretical investigation [2] has shown the existence of such a triplet state providing support to the interpretation of the susceptibility data [1]. Support for the low-lying excited triplet state has been augmented recently [3] from microscopic relaxation rate measurements for muon attached to the heme group of Oxy-Hb. We are studying by first principles Hartree-Fock procedure the energies and the electronic wave functions of the ground and triplet states and the quantitative theoretical prediction of muon magnetic hyperfine interaction in room temperature $\mu $SR measurements on Oxy-Hb. Results will be presented for hyperfine interactions of muon and other nuclei in Oxy-Hb [1] M.Cerdonio etal. Proc. Nat. Acad. Sci USA \underline {75}, 4916(1978). [2] Zalek S. Herman and Gilda H Loew JACS \underline {102}, 1815(1980).[ 3] K. Nagamine etal Proc. Jpn. Acad.Ser.B 83,120(2007).