Bulletin of the American Physical Society
2006 APS March Meeting
Monday–Friday, March 13–17, 2006; Baltimore, MD
Session P26: Focus Session: Protein Dynamics in Folding and Function |
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Sponsoring Units: DBP DCP Chair: Robert Callendar, Albert Einstein College Room: Baltimore Convention Center 323 |
Wednesday, March 15, 2006 11:15AM - 11:27AM |
P26.00001: NMR Studies of Enzyme Structure and Mechanism Invited Speaker: At least three NMR methodologies pioneered by Al Redfield, have greatly benefited enzymology: (1) the suppression of strong water signals without pre-saturation; (2) sequence specific NH/ND exchange; and (3) dynamic studies of mobile loops of proteins. Water suppression has enabled us to identify unusually short, strong H-bonds at the active sites of five enzymes (three isomerases and two esterases), and to measure their lengths from both the chemical shifts and D/H fractionation factors of the deshielded protons involved (J. Mol. Struct. 615, 163 (2002)). Backbone NH exchange studies were used to detect regions of an NTP pyrophosphohydrolase in which NH groups became selectively protected against exchange on Mg(2+) binding, and further protected on product (NMP) binding, thus locating binding sites as well as conformationally linked remote sites (Biochemistry 42, 10140 (2003)). Dynamic studies were used to elucidate the frequency of motion of a flexible loop of GDP-mannose hydrolase (66,000/sec) containing the catalytic base His-124, from exchange broadening of the side chain NH signals of His-124 in the free enzyme. The binding of Mg(2+) and GDP-mannose lock His-124 in position to deprotonate the entering water and complete the reaction. [Preview Abstract] |
Wednesday, March 15, 2006 11:27AM - 11:39AM |
P26.00002: Interference between relaxation and parameters for protein structure determination Invited Speaker: The effect of cross-correlated relaxation on scalar and dipolar coupling measurements is analyzed. We compare one-bond proton-carbon scalar and dipolar couplings of protein methine and methylene sites obtained by monitoring proton and carbon magnetization. Apparent J-coupling constants of the same pair of nuclei vary depending on the type of magnetization involved. The discrepancies are of different magnitude for methine and methylene moieties. Dynamic frequency shifts are partially responsible for the observed differences. More importantly, the largest observed variations can be explained by processes of magnetization transfer originated by cross-correlated relaxation. These later effects are not cancelled when obtaining residual dipolar couplings. [Preview Abstract] |
Wednesday, March 15, 2006 11:39AM - 11:51AM |
P26.00003: Multiple Quantum Relaxation Probes of Protein Dynamics on Multiple Timescales Ranajeet Ghose Several effects may lead to significant differences between the relaxation rates of zero-quantum coherences (ZQC) and double-quantum coherences (DQC) (collectively known as multiple-quantum coherences) generated between a pair of spin 1/2 nuclei in solution. These include the interference between the anisotropic chemical shifts of the two nuclei participating in formation of the ZQC or DQC, the individual dipolar interactions of each of the two nuclei with the same proton, and the slow modulation of the isotropic chemical shifts of the two nuclei due to conformational exchange. Motional events that occur on a timescale much faster than the rotational correlation time (picosecond-nanosecond) influence the first two effects, while the third results from processes that occur on a far slower timescale (microsecond-millisecond). An analysis of the differential relaxation of ZQC and DQC is thus informative about dynamics on the fast as well as the slow timescales. We present here a set of NMR experiments that measure the differential relaxation of ZQC and DQC involving several backbone and sidechain nuclei in proteins. These measurements provide significant insight into the complex dynamic modes that exist in the protein backbone and sidechains. A detailed understanding of these dynamic modes may provide clues into the role of dynamics in modulating protein function. [Preview Abstract] |
Wednesday, March 15, 2006 11:51AM - 12:27PM |
P26.00004: A Plastic Explosive-Degrading Enzyme Invited Speaker: The enzyme nitroreductase catalyzes reduction of high explosives such as TNT and RDX. Although a well-resolved $^{1}$H$^{15}$N-HSQC is obtained at 37 $^{\circ}$C, the HSQC at 4 $^{\circ}$C is concentrated between 7.5 and 8.5 ppm and is comprised of sharp overlapped peaks. Thus, it appears that the protein denatures upon cooling. However, the non-covalently-bound FMN cofactor is not released at the lower temperature. Similarly, ultra-violet CD spectroscopy shows that the protein retains essentially full secondary structural content at 4 $^{\circ}$C. Thus, it appears that nitroreductase exists as an ensemble of rapidly interconverting loose structures at lower temperature, only adopting a single long-lived structure above 20 $^{\circ}$C. Both saturation transfer from water and solvent proton exchange measurements, demonstrate that resonances of the poorly-dispersed spectrum represent protons closer to water, and in faster exchange with it. Thus we propose that the single well-defined structure is favored entropically, by release of water molecules that solvate the protein at 4 $^{\circ}$C. We propose that the loosely structured state plays a role in accommodating binding of diverse substrates. [Preview Abstract] |
Wednesday, March 15, 2006 12:27PM - 12:39PM |
P26.00005: Protein-Protein Interactions during Bacterial Chemotaxis using Methyl TROSY Nuclear Magnetic Resonance. Damon Hamel, Frederick Dahlquist During bacterial chemotaxis, the histidine autokinase CheA interacts with the chemotaxis receptors with the help of the coupling protein CheW. The CheA-CheW interaction is typical of many macromolecular complexes where protein-protein interactions play an important role. In this case a relatively small protein, CheW (18 kDalton), becomes part of a much larger complex. Here we describe a new method to map the residues at a protein-protein interface for macromolecular complexes of molecular weight greater than 100 kDalton. The method exploits the C13 methyl TROSY methodology developed in Lewis Kay's laboratory. The essence of the Kay approach is that a portion of the intensity of HMQC spectra of individual -(13)CH3 resonances in an otherwise deuterated macromolecule have much reduced dipole-dipole relaxation and remain sharp and relatively easy to detect , even in macromolecules of molecular mass 100 kD or greater. The reduction in dipolar interactions is lost if a given methyl group comes in close contact with other protons such as those supplied by the interface of a protonated interaction partner. Comparing the -(13)CH3 resonances of a protein of interest in the presence of a protonated versus deuterated interaction partner allows the methyls at the interface can be identified. The application of the approach for establishing points of contact between CheA and CheW will be discussed. [Preview Abstract] |
Wednesday, March 15, 2006 12:39PM - 12:51PM |
P26.00006: Structural Basis for Specific Membrane Targeting by the HIV-1 Gag Protein. Invited Speaker: In HIV-1 infected cells, newly synthesized retroviral Gag polyproteins are directed to specific cellular membranes where they assemble and bud to form immature virions. Membrane binding is mediated by Gag's matrix (MA) domain, a 132-residue polypeptide containing an N-terminal myristyl group that can adopt sequestered and exposed conformations. Membane specificity was recently shown to be regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), a cellular factor abundant in the inner leaflet of the plasma membrane (PM). We now show that phosphoinositides, including soluble analogs of PI(4,5)P2 with truncated lipids, bind HIV-1 MA and trigger myristate exposure. The phosphoinositol moiety and one of the fatty acid tails binds to a cleft on the surface of the protein. The other fatty acid chain of PI(4,5)P2 and the exposed myristyl group of MA bracket a conserved basic surface patch implicated in membrane binding. Our findings indicate that PI(4,5)P2 acts as both a trigger of the myristyl switch and as a membrane anchor, and suggest a structure-based mechanism for the specific targeting HIV-1 Gag to PI(4,5)P2-enriched membranes. [Preview Abstract] |
Wednesday, March 15, 2006 12:51PM - 1:03PM |
P26.00007: Vinculin Tail Dimerization and Paxillin Binding Invited Speaker: Vinculin is a highly conserved cytoskeletal protein that is essential for regulation of cell morphology and migration, and is a critical component of both cell-cell and cell-matrix complexes. The tail domain of vinculin (Vt) was crystallized as a homodimer and is believed to bind F-actin as a dimer. We have characterized Vt dimerization by Nuclear Magnetic Resonance (NMR) Spectroscopy and identified the dimer interface in solution by chemical shift perturbation. The Vt dimer interface in solution is similar to the crystallographic dimer interface. Interestingly, the Vt dimer interface determined by NMR partially overlaps the paxillin binding region previously defined coarsely by deletion mutagenesis and gel-blot assays. To further characterize the paxillin binding site in Vt and probe relationship between paxillin binding and dimerization, we conducted chemical shift perturbations experiments using a paxillin derived peptide, LD2. Our NMR experiments have confirmed that the paxillin binding site and the Vt dimerization site partially overlap, and we have further characterized both of these two binding interfaces. Information derived from these studies was used to identify mutations in Vt that selectively perturb paxillin binding and Vt self-association. These mutants are currently being characterized for their utility in structural and biological analyses to elucidate the role of paxillin binding and Vt dimerization in vinculin function. [Preview Abstract] |
Wednesday, March 15, 2006 1:03PM - 1:15PM |
P26.00008: Protein dynamics and allostery Babis Kalodimos Cooperativity and allostery are phenomena of universal importance in biological systems. According to the classical ``mechanical'' view allosteric interactions are mediated by a series of discrete changes in bonding interactions that alter the protein conformation. Nevertheless, proteins may have adopted additional mechanisms for energetically linking distant sites, thereby allowing a signal to be propagated over long distances. We have identified a cooperative biological system wherein allosteric interactions appear to be mediated exclusively by transmitted changes in protein motions. Changes in the structure, fast and slow protein motions and the redistribution of the native-state ensemble along the cooperative reaction coordinate have been characterized. [Preview Abstract] |
Wednesday, March 15, 2006 1:15PM - 1:27PM |
P26.00009: Protein Dynamics in an RNA Binding Protein Invited Speaker: Using $^{15}$N NMR relaxation measurements, analyzed with the Lipari-Szabo formalism, we have found that the human U1A RNA binding protein has ps-ns motions in those loops that make contact with RNA. Specific mutations can alter the extent and pattern of motions, and those proteins inevitably lose RNA binding affinity. Proteins with enhanced mobility of loops and termini presumably lose affinity due to increased conformational sampling by those parts of the protein that interact directly with RNA. There is an entropic penalty associated with locking down those elements upon RNA binding, in addition to a loss of binding efficiency caused by the increased number of conformations adopted by the protein. However, in addition to local conformational heterogeneity, analysis of molecular dynamics trajectories by Reorientational Eigenmode Dynamics reveals that loops of the wild type protein undergo correlated motions that link distal sites across the binding surface. Mutations that disrupt correlated motions result in weaker RNA binding, implying that there is a network of interactions across the surface of the protein. (KBH was a Postdoctoral Fellow with Al Redfield from 1985-1990). This work was supported by the NIH (to KBH) and NSF (SAS). [Preview Abstract] |
Wednesday, March 15, 2006 1:27PM - 1:39PM |
P26.00010: Axial Rotation of Lipids in Membranes Invited Speaker: This study was motivated by Mary Roberts and Al Redfield, who proposed that the observed 10 ns decay time in their phosphorous NMR measurements of DPPC in bilayers originated from axial rotation of the lipid. Analyses of correlation functions and average first passage times from 50 ns molecular dynamics of DPPC bilayers strongly support their interpretation. The rotational anisotropy of a lipid in a bilayer is close to 1.0, in contrast to the 2.5 expected for a hydrodynamic cylinder with lipid dimensions. This implies that axial rotation is dominated by headgroup, not tail, interactions. [Preview Abstract] |
Wednesday, March 15, 2006 1:39PM - 2:15PM |
P26.00011: High Resolution Field Cycling NMR in Biopolymers in Solution: Current and Potential Applications. Invited Speaker: I have been exploring the feasibility and utility of performing high resolution relaxation and cross-relaxation (NOE) in an unmodified shared commercial NMR instrument (PNAS 101:17066-17071) in collaboration with Mary Roberts of Boston College and Elan Eisenmesser of Brandeis. We can move a sample from 11.7 T to low (fringe) field 40- 80 cm above the commercial probe, and back, in 0.3 to 0.5 sec. I am making the system move the sample more gently, to avoid protein denaturation, using a stepping-motor timing-belt linear-motor. I willreview our initial papers on a DNA octamer, and phospholipidvesicles using phosphorus NMR. We emphasize phosphorus,in part, because relaxation studies over a wide range can make behavior of this biologically important species more readily interpretable than at high field alone. Then I will discuss a range of biochemical experiments, not yet done, using the full capability of our commercial instrument for multidimensional preparation and detection before and after field-cycling, and utilizing other nuclear species (H, C, and N). These involve especially use of electron-paramagnetic species and/or studies of small molecules in fast binding exchange with larger ones, and/or increased hetero-NOE effects at low field, to get new information on dynamics and structural preferences. Predictably we have found that, once we start to work on a specific biopolymer problem, we think of more things to do than we expected at first. [Preview Abstract] |
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