### Session P39: Physics of Proteins IV: Folding, Dynamics and Function

 Wednesday, March 23, 2011 8:00AM - 8:12AM P39.00001: Competition between chemical denaturation and macromolecular crowding effects on the folding dynamics of proteins Antonios Samiotakis , Margaret Cheung It is well known that proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been established that the so-called macromolecular crowding'' effect can enhance the folding stability of proteins by destabilizing their unfolded states. On the other hand, chemical and thermal denaturation are often used in experiments as tools to destabilize protein structures when probing a protein's folding landscape. However, little is known about the combined effects of these competing phenomena on proteins. In this work, we use coarse-grained molecular simulations to study the thermodynamic and kinetic properties of the small peptide Trp-cage, in the combined presence of macromolecular crowders and chemical denaturant. With the use of an energy function derived by all-atomistic simulations in the presence of urea, we investigate the thermodynamics and kinetics of Trp-cage's folding mechanism at several concentrations of urea. The effects of the competition between stabilization by macromolecular crowding and destabilization by chemical denaturation will also be discussed. Wednesday, March 23, 2011 8:12AM - 8:24AM P39.00002: Can understanding the packing of side chains improve the design of protein-protein interactions? Alice Zhou , Corey O'Hern , Lynne Regan With the long-term goal to improve the design of protein-protein interactions, we have begun extensive computational studies to understand how side-chains of key residues of binding partners geometrically fit together at protein-peptide interfaces, e.g. the tetratrico-peptide repeat protein and its cognate peptide). We describe simple atomic-scale models of hydrophobic dipeptides, which include hard-core repulsion, bond length and angle constraints, and Van der Waals attraction. By completely enumerating all minimal energy structures in these systems, we are able to reproduce important features of the probability distributions of side chain dihedral angles of hydrophic residues in the protein data bank. These results are the crucial first step in developing computational models that can predict the side chain conformations of residues at protein-peptide interfaces. Wednesday, March 23, 2011 8:24AM - 8:36AM P39.00003: Insight into the mechanics of the selectivity filter of \textit{Escherichia coli} aquaporin Z Guodong Hu , L.Y. Chen Aquaporin Z (AQPZ) is a tetrameric protein that forms water channels in Escherichia coli's cell membrane. The histinine residue in the selectivity filter (SF) region plays an important role in the transport of water across the membrane. In this work, we perform equilibrium molecular dynamics (MD) simulation to illustrate influences of two different protonation states and the gate mechanics of the SF. We calculate the pore radii in the SF region versus the simulation time. We perform steered MD to compute the free energy profile, i.e., the potential of the mean force (PMF) a water molecule through the SF region. We calculate the binding energy of one water molecule with the SF region residues, using Gaussian. The hydrogen bonds formed between the side chains of Hsd 174 and side chains of Arg189 play important roles in the selectivity filter mechanics of AQPZ. The radii of the pores, hydrogen bond analysis, and free energies show that Hsd is favored than Hse. Wednesday, March 23, 2011 8:36AM - 8:48AM P39.00004: The Effect of Phosphate Buffered Saline (1x PBS) on Induced Thermal Unfolding and Low Frequency Dielectric Spectra of Lysozyme Klaida Kashuri , Hektor Kashuri , Germano Iannacchione It is well known that the folding / unfolding of proteins is related directly to their structure and functionality. Calorimetry (both AC and MDSC) studies as well as low-frequency (1Hz to 100 kHz) dielectric measurements have been performed on hen egg white lysozyme dissolved in PBS (pH 7.4) from 20 to 100 $^{\circ}$C. From the heat capacity profile, the temperatures and related an enthalpy change of the protein denaturing is probed. The heat capacity peak broadens and new features are reveled as the temperature scan rate is lowered to +0.017 K/min for the AC calorimetric method. Significant differences are observed using the (M)DSC technique at scan rates of from 1 to 5 K/min. The temperature dependence of the permittivity, $\epsilon '$, and the loss factor, $\epsilon ''$, at 100 kHz of the diluted protein show features associated with those seen in the heat capacity (AC and MDSC). All results are interpreted in terms of protein denaturing then subsequent gelation that depend on protein sample concentration, which is supported by the frequency dependence of the permittivity at room temperature after thermally cycling Wednesday, March 23, 2011 8:48AM - 9:00AM P39.00005: The effect of macromolecular crowding, ionic strength and calcium binding on calmodulin dynamics Qian Wang , Kao-Chen Liang , Neal Waxham , Margaret Cheung The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM in response to the changing intracellular environment, we use a combined method of computer simulation and experiments based on circular dichroism (CD). The conformation, helicity and EF hand orientation of CaM are analyzed computationally to address the effect of macromolecular crowding, ionic strength and calcium binding in the experiments. We applied a unique solution of charges computed from QM/MM to accurately represent the charge distribution in the transition from apo-CaM to holo-CaM. Computationally, we found that a high level of macromolecular crowding, in addition to calcium binding and ionic strength, can impact the conformation, helicity and the EF hand orientation of CaM. Our result may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells. Wednesday, March 23, 2011 9:00AM - 9:12AM P39.00006: $\alpha$-Helical to $\beta$-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein Jesse Singh , Paul Whitford , Natha Hayre , Daniel Cox , Jos\'e Onuchic We employ all-atom structure-based models with mixed basis contact maps to explore whether there are any significant geometric or energetic constraints limiting conjectured conformational transitions between the alpha-helical ($\alpha$H) and the left handed beta helical (LHBH) conformations for the C-terminal (residues 166-226) of the mammalian prion protein. The LHBH structure has been proposed to describe infectious oligomers and one class of in vitro grown fibrils, as well as possibly self- templating the conversion of normal cellular prion protein to the infectious form. Our results confirm that the kinetics of the conformation change are not strongely limited by large scale geometry modification and there exists an overall preference for the LHBH conformation. Wednesday, March 23, 2011 9:12AM - 9:24AM P39.00007: Dynamics of Protein Carbonmonoxyhemoglobin on Multiple Length Scales Jyotsana Lal , Robert Fischetti , Lee Makowski , Peter Fouquet , Marco Maccarini , Nancy Ho , Chien Ho A combination of wide-angle x-ray solution scattering (WAXS) and neutron spin echo spectroscopy (NSE) was used to probe the structure and dynamics of carbonmonoxy hemoglobin (HbCO) in the presence and absence of the allosteric effector inositol hexaphosphate (IHP). IHP shifts the structure of HbCO slightly towards an unliganded, (deoxy)-state conformation. Two potential binding sites for IHP are consistent with the WAXS data, one near each end of the central channel. IHP binding slows the self-correlation times of some protons, most likely those immediately adjacent to the bound IHP, and simultaneously induces an increase in the relaxation rate of correlated motions with length scales comparable to the $\alpha \beta$-dimer. IHP binding increases the spatial extent of these fluctuations by about 20{\%}. This suggests that when hemoglobin binds CO, its conformation is confined to a relatively narrow structural ensemble residing within a functionally well defined energy well. On the other hand, when it binds both CO and IHP, in response to the contradictory stresses applied by these two ligands, it adopts an incommensurate structure with a conformation exploring a broad structural ensemble. Wednesday, March 23, 2011 9:24AM - 9:36AM P39.00008: Infrared Spectroscopy Measurements of Protein Dynamics and Mechanism Curtis W. Meuse , Joseph B. Hubbard Infrared spectroscopy has long been used to deduce the concentration and secondary structures of proteins in a variety of static and time resolved applications. Our focus is on developing new infrared methods to compare the structure, dynamics and function of nearly identical protein samples, in different environments, to apply to the problem of identifying bio-similar protein therapeutics. We have developed an order parameter describing protein conformation variations around the average molecular values. By comparing our order parameter and amide hydrogen/deuterium exchange methods, we explore the relationship between protein stability and the dynamics of the protein conformational distribution. Examples include lysozyme and albumin in solution, cytochrome c interacting with lipid membranes of varying net-negative surface charge density, fibrinogen on different polymer surfaces and bacteriorhodopsin during its photocycle. Wednesday, March 23, 2011 9:36AM - 9:48AM P39.00009: Comparing potential copper chelation mechanisms in Parkinson's disease protein Frisco Rose , Miroslav Hodak , Jerry Bernholc We have implemented the nudged elastic band (NEB) as a guided dynamics framework for our real-space multigrid method of DFT-based quantum simulations. This highly parallel approach resolves a minimum energy pathway (MEP) on the energy hypersurface by relaxing intermediates in a chain-of-states. As an initial application we present an investigation of chelating agents acting on copper ion bound to $\alpha$-synuclein, whose misfolding is implicated in Parkinson's disease (PD). Copper ions are known to act as highly effective misfolding agents in a-synuclein and are thus an important target in understanding PD. Furthermore, chelation therapy has shown promise in the treatment of Alzheimer's and other neuro-degenerative diseases with similar metal-correlated pathologies. At present, our candidate chelating agents include nicotine, curcumin and clioquinol. We examine their MEP activation barriers in the context of a PD onset mechanism to assess the viability of various chelators for PD remediation. Wednesday, March 23, 2011 9:48AM - 10:00AM P39.00010: Investigating hexameric helicases: Single-molecule studies of DnaB and T4 gp41 Omar Saleh , Noah Ribeck , John Berezney Hexameric, ring-shaped motor proteins serve as replicative helicases in many systems. They function by encircling and translocating along ssDNA, denaturing dsDNA in advance of its motion by sterically occluding the complementary strand to the outside of the ring. We investigate the helicase activity of two such motors using single-molecule measurements with magnetic tweezers. First, we measure the activity of the \textit{E. coli} helicase DnaB complexed with the tau subunit of the Pol III holoenzyme. Tau is known from bulk measurements to stimulate DnaB activity (Kim et al., \textit{Cell}, 1996); we investigate the means of this stimulation. Second, we measure helicase activity of the T4 phage helicase gp41 in multiple tethered DNA geometries. Previous work on DnaB showed a dependence of helicase activity on DNA geometry (Ribeck et al., \textit{Biophys. J}., 2010); here, we test gp41 for similar behavior to see whether it is a common characteristic of hexameric helicases. Wednesday, March 23, 2011 10:00AM - 10:12AM P39.00011: Copper attachment to prion protein at a non-octarepeat site Miroslav Hodak , Jerry Bernholc Prion protein (PrP) plays a causative role in a group of neurodegenerative diseases, which include mad cow disease'' or its human form variant Creutzfeld-Jacob disease. Normal function of PrP remains unknown, but it is now well established that PrP can efficiently bind copper ions and this ability has been linked to its function. The primary binding sites are located in the so-called octarepeat region located between residues 60-91. While these are by now well characterized, the sites located outside these region remain mostly undetermined. In this work, we investigate the properties of Cu binding site located at His 111 using recently developed hybrid Kohn-Sham/orbital-free density functional simulations. Experimental data indicate that copper is coordinated by either four nitrogens or three nitrogens and one oxygen. We investigate both possibilities, comparing their energetics and attachment geometries. Similarities and differences with other binding sites and implications for PrP function will also be discussed. Wednesday, March 23, 2011 10:12AM - 10:24AM P39.00012: Pressure and Temperature Effects on Polypeptides and Biomolecules Probed by Micro-Raman Spectroscopy Sanghoon Park , Alfons Schulte We investigate pressure and temperature effects on the secondary structure of Poly-L-glutamic acid (PGA) in D$_{2}$O buffer (pH 5.4) solution. Our setup employs a Raman microscope equipped with a micro-capillary high-pressure cell and a variable temperature stage. Raman spectra are acquired over the pressure range from 0.1 to 300 MPa while the temperature can be varied from 270 K to 330 K. The amide I band of PGA is sensitive to pressure and temperature, and by spectral deconvolution we determine the relative contributions due to $\alpha$-helix and random coil conformations. The amount of $\alpha$-helix increases with increasing pressure. Extensions of these experiments to model proteins and lipids are presented.