Bulletin of the American Physical Society
2007 APS March Meeting
Volume 52, Number 1
Monday–Friday, March 5–9, 2007; Denver, Colorado
Session N35: Focus Session: Time Resolved Structural Investigations on Protein Folding and Function |
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Sponsoring Units: DBP DCP Chair: Aihua Xie, Oklahoma State University Room: Colorado Convention Center 405 |
Wednesday, March 7, 2007 8:00AM - 8:36AM |
N35.00001: Correlating folding and signaling in a photoreceptor by single molecule measurements and energy landscape calculations Invited Speaker: Receptor activation is a fundamental process in biological signaling. We study the structural changes during activation of photoactive yellow protein (PYP). This is triggered by photoisomerization of the p-coumaric acid (pCA) chromophore of PYP, which converts the initial pG state into the activated pB state. Mechanical unfolding of Cys-linked PYP multimers probed by atomic force microscopy (AFM) in the presence and absence of illumination reveals that the core of the protein is extended by 3 nm and destabilized by 30 percent in pB. These results establish a generally applicable single molecule approach for mapping functional conformational changes to selected regions of a protein and indicate that stimulus-induced partial protein unfolding can be employed as a signaling mechanism. Comparative measurements, Jarzynski-Hummer-Szabo analysis of the data, and steered MD simulations of two double-Cys PYP mutants reveal strong anisotropy in the unfolding mechanism along the two axes defined by the Cys residues. Unfolding along one axis exhibits a transition-state-like feature where six hydrogen bonds break simultaneously. The other axis displays an unpeaked force profile reflecting a non-cooperative transition, challenging the notion that cooperative unfolding is a universal feature in protein stability. MD simulations with a coarse-grained protein model show that the folding of pG is two-state, consistent with experimental observations. In contrast, the folding free energy surface of a coarse-grained model of pB involves an on-pathway partially unfolded intermediate that closely matches experimental data. The results reveal that interactions between the pCA and its binding pocket can switch the energy landscape for PYP from two- to three-state folding, and show how this can be exploited to trigger large functionally important protein conformational changes. [Preview Abstract] |
Wednesday, March 7, 2007 8:36AM - 8:48AM |
N35.00002: Spectroscopic probes of enzyme-ligand interaction dynamics Christopher Cheatum, Jigar Bandaria, Samrat Dutta, Sarah Hill, Amnon Kohen Formate dehydrogenase catalyzes the NAD-dependent oxidation of formate to carbon dioxide. The intrinsic chemical step involves hydride transfer from formate to the nicotinamide ring of NAD. As with several other NAD-dependent dehydrogenases, kinetic measurements suggest that thermal fluctuations of the enzyme are important in the hydride-transfer reaction. We have measured the dynamics of enzyme-inhibitor interactions in binary and ternary complexes of formate dehydrogenase with pseudohalides using infrared photon-echo spectroscopy. The pseudohalides are excellent vibrational chromophores that are known to be sensitive reporters of interactions with their local environments. They are also excellent inhibitors for formate dehydrogenase. Our measurements reveal significant differences in the dynamics of the different binary and ternary complexes. By comparing and contrasting the dynamics for different complexes we gain insight into the active-site components that make the most important contributions to the observed dynamics. [Preview Abstract] |
Wednesday, March 7, 2007 8:48AM - 9:00AM |
N35.00003: Shallow Free Energy Landscapes Remodelled by Ligand Binding Troy Messina, David Talaga, Emilio Gallichio, Ronald Levy Glucose/galactose binding protein (GGBP) functions as part of a larger system of proteins for molecular recognition and signalling in enteric bacteria. Here we report on the thermodynamics of conformational equilibrium distributions of GGBP 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 GGBP has with cytoplasmic membrane proteins. [Preview Abstract] |
Wednesday, March 7, 2007 9:00AM - 9:12AM |
N35.00004: High-throughput biophysics of functional tuning in photoactive yellow protein Wouter Hoff, Andrew Philip, George Papadantonakis The relationship between the structure of a protein and its function is a central unresolved problem in biology. We use photoactive yellow protein (PYP) to develop quantitative high-throughput methods to study this problem. PYP is a small bacterial photoreceptor with rhodopsin-like photochemistry based on its p-coumaric acid (pCA) chromophore. The absorbance maximum and pKa of the pCA in the active site of native PYP are shifted from 400 nm and 9.0 in water to 446 nm and 2.8 in the protein. Thus, PYP offers a unique model system to probe protein-ligand interactions. Here we show that high-throughput microscale methods can be used for quantitative biophysical studies of the absorbance spectrum PYP, its fluorescence quantum yield, apparent pKa of the pCA, protein stability against chemical denaturation, and kinetics of the last PYP photocycle step. A wide range of properties was observed among the mutants, and structural features that tune functional properties were identified. These results open the way for high-throughput quantitative biophysical studies of PYP. [Preview Abstract] |
Wednesday, March 7, 2007 9:12AM - 9:24AM |
N35.00005: Probing protein dynamics using Fluorescence Resonance Energy Transfer with donors of different lifetimes Weiqun Peng, Tania Chakrabarty, Paul Goldbart, Paul Selvin Fluorescence resonance energy transfer (FRET), using nanosecond dyes, and its derivative, Lanthanide-based resonance energy transfer (LRET), using millisecond-lifetime lanthanide chelates, are methods to measure distances on the 2-10 nm length-scale. It has been found that in certain systems energy transfer efficiency E for FRET and LRET measurements can be dramatically different [Chakrabarty et al., PNAS, 99: 6011-6016 (2002)]. Here we develop a theoretical model that shows that the dramatic difference can be explained by the presence of intrinsic dynamics of the system. Furthermore, we quantitatively investigate how information about the time-scale and distance-scale associated with the intrinsic dynamics can be inferred, by comparison of FRET and LRET results. [Preview Abstract] |
Wednesday, March 7, 2007 9:24AM - 10:00AM |
N35.00006: Advanced Infrared Spectroscopy for Time-Resolved Structural Investigation of Protein Structure and Function Invited Speaker: The human genome encodes approximately 30,000 different proteins. A single mutation at a critical site of one protein can cause serious diseases, such as cardiac failure and cancer. This illustrates the significant role of protein structures in protein functions. In order to obtain a fundamental understanding of protein structure-function relation, we must develop and employ both physical theories and experimental techniques. In my talk, I will report both experimental and computational studies on vibrational structural markers for advanced infrared spectroscopy, slaved protein structural dynamics, and ``electrostatic epicenter'' model as a general mechanism for activation of receptor proteins in cell signaling. [Preview Abstract] |
Wednesday, March 7, 2007 10:00AM - 10:12AM |
N35.00007: Channel noise reduction due to gating charge effects Gerhard Schmid, Igor Goychuk, Peter H\"anggi We investigate the influence of gating charge effects on the channel noise-induced spontaneous spiking activity of excitable membrane patches [1] within a stochastic Hodgkin-Huxley model [2]. The random switching of the channel gates between an open and a closed configuration is always connected with movement of gating charge within the cell membrane. At the beginning of an action potential the gating current is opposite to the direction of the ion current through the membrane. Therefore, the excitability is expected to become reduced due to the influence of gating current. Our study revealed that while the deterministic modelling with gating charge effects does not differ dramatically from the original Hodgkin-Huxley model for the standard set of parameters, the corresponding stochastic model which takes into account the channel noise -- i.e. the fluctuations of the number of open ion channels -- does behave very differently for intermediate-to-large membrane patch sizes. A main finding is that spontaneous spiking activity becomes drastically reduced [1]. \newline \noindent [1] G. Schmid, I. Goychuk, and P. H\"anggi, Phys. Biol., in press (2006); (arXiv:abs/q-bio.NC/0611040). \newline [2] G. Schmid, I. Goychuk, and P. H\"anggi, Europhys. Lett. {\bf 56}, 22 (2001) [Preview Abstract] |
Wednesday, March 7, 2007 10:12AM - 10:24AM |
N35.00008: A Molecular Dynamics-Decorated Finite Element Method (MDeFEM) Framework for Simulating the Gating of Mechanosensitive Channels Xi Chen, Yuye Tang, Guoxin Cao, Jejoong Yoo, Arun Yethiraj, Qiang Cui The gating pathways of mechanosensitive channels of large conductance (MscL) are studied using the finite element method. The phenomenological model treats transmembrane helices as elastic rods and the lipid membrane as an elastic sheet of finite thickness. The interactions between various continuum components are derived from atomistic energy calculations. The structural variations along the gating pathway are consistent with previous analyses based on structural models and biased molecular-dynamics simulations. Upon membrane bending, there is notable and nonmonotonic variation in the pore radius. This emphasizes that the gating behavior of MscL depends critically on the form of the mechanical perturbation. Compared to popular all-atom simulations, the MDeFEM framework offers a unique alternative to bridge detailed intermolecular interactions and biological processes occurring at large spatial and timescales. It is envisioned that such a hierarchical multiscale framework will find great value in the study of a variety of biological processes involving complex mechanical deformations such as muscle contraction and mechanotransduction. [Preview Abstract] |
Wednesday, March 7, 2007 10:24AM - 10:36AM |
N35.00009: Develop vibrational structural makers for probing the protonation state and hydrogen bonding interactions of tyrosine in proteins and their functional intermediates Anupama Thubagere, Beining Nie, Edward Manda, Aihua Xie Proteins are dynamic in nature. In order to understand how a protein performs its function based on laws of physics, it is critical to probe and investigate functionally important structural transitions of the protein. Time-resolved infrared spectroscopy offers excellent time resolution (picoseconds to seconds), and contains extensive structural information. The real challenge is how to extract structural information from time-resolved infrared data. We will report computational methods for developing vibrational structural markers of tyrosine. Using density function theory (DFT) based first principle computational studies combined with experimental data, we found that it is possible to unambiguously determine if the phenolic ring in Tyrosine is neutral or negatively charged based on the frequency of one ring vibrational mode. In addition, we show that it possible to determine the number and nature of hydrogen bonding interactions of a phenolic group in proteins using a combination of C-O stretching and O-H stretching frequencies (2D vibrational spectroscopy). [Preview Abstract] |
Wednesday, March 7, 2007 10:36AM - 10:48AM |
N35.00010: Correlated Fluorescence Parameters of Single Molecules Claudiu Gradinaru, David Chandler, Carl Hayden A novel detection system is used in a confocal optical microscope for measuring correlated fluorescence lifetimes and spectra. Fluorescence photons emitted from a sample are imaged through a dispersive optical system onto a time- and position-sensitive detector. For each photon the apparatus records the wavelength, the emission time relative to the laser excitation pulse and the absolute detection time, so that correlations among all the fluorescence properties are maintained. A histogram over many photons can generate a full fluorescence spectrum and a correlated decay plot at every pixel in a fluorescence image. The complex data structure allows mapping the time-dependent distribution of multiple fluorescent species in a sample and enables monitoring the dynamics of single molecules on a time scale that spans from picoseconds to minutes. Unique correlations between intensity, spectrum and lifetime prove useful for tracking changes in the nanoenvironment of fluorescent probes. The detection method also provides a more complete description of the fluorescence resonance energy transfer (FRET) than conventional microscopy techniques, as demonstrated by single-pair FRET experiments between dyes spaced apart by short peptides and by dsDNA chains. [Preview Abstract] |
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