Side-chain Fluctuations Research Articles

A Multifrequency Electron Spin Resonance Study of T4 Lysozyme Dynamics Using the Slowly Relaxing Local Structure Model

Electron spin resonance (ESR) spectra were obtained at 250 and 9 GHz for nitroxide-labeled mutants of the protein T4 lysozyme in aqueous solution over a range of temperatures from 2 to 37.5 °C. Two mutants labeled at sites 72 and 131 were studied and compared. The mutant sites are solvent exposed and free of tertiary interactions with other side chains, but the former is at the center of a 5 turn helix, whereas the latter site is on a small two and a half turn helix. The 250 GHz ESR spectra, because of their “fast time scale”, are rather insensitive to the slow overall tumbling motion of the protein. Thus, they are qualitatively different for the two mutants, implying that there are different local dynamics at the two sites. The 9 GHz spectra, which are significantly affected by the overall tumbling and are less sensitive to the internal dynamics, do not show such marked differences between the two sites. The 250 and 9 GHz spectra for each mutant and temperature were simultaneously fit to the slowly relaxing local structure (SRLS) model for slow-motional ESR, using newly developed software. The SRLS model explicitly accounts for the overall tumbling of the protein and the internal modes of motion, which include the motion of the nitroxide side chain (expected to be the same for both mutants) and backbone fluctuations. Very good simultaneous fits are obtained. Whereas two conformers (or spectral components) are typically detected at the lower temperatures, only a single component is observed at the higher temperatures. The significant differences in the high-frequency spectra for the two mutants are readily attributed mainly to a difference in their respective local ordering. That is, site 72 exhibits significantly greater local ordering than does site 131, which is expected from the greater rigidity of the larger helix on which the 72 site is located. The results of this multifrequency study are compared with a previous 9 GHz study. A description of the application of the SRLS model in such a multifrequency study is provided.

Functionality maps of the ATP binding site of DNA gyrase B: generation of a consensus model of ligand binding.

The Multiple Copy Simultaneous Search method (MCSS) was used to construct consensus functionality maps for functional group binding in the ATP binding site of DNA gyrase B. To account for the conformational flexibility of the protein active site, which involves small side chain fluctuations as well as large-scale loop motions, the calculations were done for three different conformations of the 24 kDa subdomain of DNA gyrase B. A postprocessing procedure that employs a continuum dielectric model to include solvent effects was used to calculate the binding free energy for every functional group. These results were ranked according to their affinity for DNA gyrase B and clustered using a new procedure based on van der Waals contacts that is better adapted for cases where multiple conformations are being considered. A total of 23 different functional groups were tested. The results gave consensus maps that indicate those functional group binding sites that are insensitive to the specific protein conformation. The maps also demonstrate that functional groups other than those found in the known ligands may bind competitively in the binding sites of known ligands. This suggests numerous scaffolds that can be used in the development of new ligands for the ATP and coumarinic binding sites in DNA gyrase B. Finally, the calculations show the existence of alternative binding sites near the known binding sites that could be targeted in the rational design for new inhibitors.

Protein side-chain motion and hydration in proton-transfer pathways. Results for cytochrome p450cam.

Proton-transfer reactions form an integral part of bioenergetics and enzymatic catalysis. The identification of proton-conducting pathways inside a protein is a key to understanding the mechanisms of biomolecular proton transfer. Proton pathways are modeled here as hydrogen bonded networks of proton-conducting groups, including proton-exchanging groups of amino acid side chains and bound water molecules. We focus on the identification of potential proton-conducting pathways inside a protein of known structure. However, consideration of the static structure alone is often not sufficient to detect suitable proton-transfer paths, leading, for example, from the protein surface to the active site buried inside the protein. We include dynamic fluctuations of amino acid side chains and water molecules into our analysis. To illustrate the method, proton transfer into the active site of cytochrome P450cam is studied. The cooperative rotation of amino acids and motion of water molecules are found to connect the protein surface to the molecular oxygen. Our observations emphasize the intrinsic dynamical nature of proton pathways where critical connections in the network may be transiently provided by mobile groups.

Spectroscopic and molecular dynamics simulation studies of the interaction of insulin with glucose

The interaction between monomeric insulin and monosaccharides has been investigated through circular dichroism, fluorescence spectroscopy and two dimensional nuclear magnetic resonance. CD spectra indicate that d-glucose interacts with monomeric insulin whereas d-galactose, d-mannose and 2-deoxy- d-glucose have a lower effect. Fluorescence emission was quenched at sugar concentrations of 5–10 mM. Titration with the different sugars produces a quenching of the tyrosine spectrum from which a binding free energy value for the insulin–sugar complexes has been evaluated. Transfer nuclear Overhauser enhancement NMR experiments indicate the existence of dipolar interactions at short interatomic distances between C-1 proton of d-glucose in the β form and the monomeric insulin. Further, NMR total correlation spectra experiments revealed that the hormone is in the monomeric form and that upon addition of glucose no aggregation occurs. The interaction does not involve relevant changes in the secondary structure of insulin suggesting that the interaction occur at the side chain level. Molecular dynamics simulations and modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additional informations to locate the putative binding sites of d-glucose to insulin.

Spectroscopic Ellipsometry and Fluorescence Study of Thermochromism in an Ultrathin Poly(diacetylene) Film: Reversibility and Transition Kinetics

We have investigated the thermochromic transition of an ultrathin poly(diacetylene) film. The Langmuir film is composed of three layers of polymerized 10,12-pentacosadiynoic acid [CH{sub 3}(CH{sub 2}){sub 11}C{triple_bond}CC{triple_bond}C(CH{sub 2}){sub 8}COOH] (poly-PCDA) organized into crystalline domains on a silicon substrate. Spectroscopic ellipsometry and fluorescence intensity measurements are obtained with in-situ temperature control. Poly-PCDA films exhibit a reversible thermal transition between the initial blue form and an intermediate ''purple'' form that exists only at elevated temperature (between 303-333 K), followed by an irreversible transition to the red form after annealing above 320 K. We propose that the purple form is thermally distorted blue poly-PCDA, and may represent a transitional configuration in the irreversible conversion to red. This hypothesis is supported by the appearance of unique features in the absorption spectra for each form as derived from the ellipsometry measurements. Significant fluorescence emission occurs only with the red form, and is reduced at elevated temperatures while the absorption remains unchanged. Reduced emission is likely related to thermal fluctuations of the hydrocarbon side chains. Time-resolved fluorescence measurements of the irreversible transition have been performed. Using a first-order kinetic analysis of these measurements we deduce an energy barrier of 17.6 {+-} 1.1 kcal mol{sup -1} between the blue and red forms.

Effects of ligand binding on the internal dynamics of maltose-binding protein.

Ligand binding to proteins often causes large conformational changes. A typical example is maltose-binding protein (MBP), a member of the family of periplasmic binding proteins of Gram-negative bacteria. Upon binding of maltose, MBP undergoes a large structural change that closes the binding cleft, i.e. the distance between its two domains decreases. In contrast, binding of the larger, nonphysiological ligand beta-cyclodextrin does not result in closure of the binding cleft. We have investigated the dynamic properties of MBP in its different states using time-resolved tryptophan fluorescence anisotropy. We found that the 'empty' protein exhibits strong internal fluctuations that almost vanish upon ligand binding. The measured relaxation times corresponding to internal fluctuations can be interpreted as originating from two types of motion: wobbling of tryptophan side-chains relative to the protein backbone, and orientational fluctuations of entire domains. After binding of a ligand, domain motions are no longer detectable and the fluctuations of some of the tryptophan side-chains become rather restricted. This transformation into a more rigid state is observed upon binding of both ligands, maltose and the larger beta-cyclodextrin. The fluctuations of tryptophan side-chains in direct contact with the ligand, however, are affected in a slightly different way by the two ligands.

Structural Dynamics of Ligand Diffusion in the Protein Matrix: A Study on a New Myoglobin Mutant Y(B10) Q(E7) R(E10)

A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) → Tyr, His(E7) → Gln, and Thr(E10) → Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O 2, and CO from within the protein (in a “geminate” mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O 2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O 2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O 2 binding are reduced 10-fold. While there is no geminate recombination with O 2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major “secondary site,” located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the “primary site” identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O 2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O 2 delivery to tissue at a rate compatible with physiology.

Cooperative charge fluctuations by migrating protons in globular proteins.

A review of the hydrogen bonded network on the protein surface shows the presence of a charged complex system with parallel and competitive interactions, including ionizable side-chains, migrating protons, bound water and nearby backbone peptides. This system displays cooperative effects of dynamical nature, reviewed for lysozyme as a case. By increasing the water coverage of the protein powder, the bound water cluster exhibits a percolative transition, detectable by the onset of large water-assisted displacements of migrating protons, with a parallel emergence of protein mobility and biological function. By lowering the temperature, migrating protons exhibit a glassy dielectric relaxation in the low frequency range, pointing to a frustration by competing interactions similar to that observed in spin glasses and fragile glass forming liquids. The observation of these dissipative processes implies the occurrence of spontaneous charge fluctuations. A simplified model of the protein surface, where conformational and ionizable side-chain fluctuations are averaged out, is used to discuss the statistical physics of these cooperative effects. Some biological implications of this dynamical cooperativity for enzymatic activity are briefly suggested at the end.

Anatomy by computer experiment of the exchange of a water molecule buried in human apolipoprotein E

NMR experiments show that even water molecules that are well ordered in a crystal structure exchange with the external solvent. Despite crucial progress on the understanding of the exchange of crystal-buried water molecules, the detailed pathways followed by a water molecule to escape from or penetrate into the protein interior are unknown. The exchange of a crystal water molecule buried in the low-density lipoprotein receptor-binding domain of human apolipoprotein E with a water molecule from the external solvent was detected and monitored in a molecular dynamics simulation. This simulation shows that the escape of the crystal water molecule from the protein interior and the penetration of the water molecule from the bulk occur by a single-pathway mechanism involving conformational fluctuations of arginine and tryptophan sidechains. Along the pathway the exchanging water molecule interacts specifically with protein atoms by way of a varying pattern of hydrogen bonds. The exchange pathway revealed by the molecular dynamics trajectory suggests a mechanism by which hydrogen bonds work in relay to permit either the penetration or the expulsion of a water molecule. This result may have important implications not only on the process of water exchange but also to probe ligand binding to proteins.

Optical noise and dynamical properties of liquid crystal comb polymers with different mesogenic groups

The light scattered by a comb polymer with a polyacrylamide main chain and biphenyl-based mesogenic units containing the substituent R CN in the biphenyl core is characterized by a stationary noise which has been measured between 70 C and 140 C in the frequency interval 5 10 - 2 nu 10Hz. This optical noise is analysed by means of a specific procedure introduced to clarify the role of random movements of segments of the main chains on the side chain fluctuations. The RMS amplitude of the random molecular movements, the relaxation times of the side chain and main chain fluctuations, and the damping coefficients governing their motion are investigated as functions of temperature. The results are compared with those recently obtained on a similar polymer characterized by R H. Common features and differences are brought into evidence and discussed in terms of the different mesophase structures exhibited by the two polymers in the temperature interval considered.

Time-resolved near UV circular dichroism and absorption studies of carbonmonoxymyoglobin photolysis intermediates

Abstract Time-resolved circular dichroism (TRCD) spectra of ligand photolysis intermediates of horse skeletal carbonmonoxymyoglobin (MbCO) in the near UV (250–300 run) region are obtained to probe the environment of protein aromatic side chains as a function of time. Since X-ray data reveal compact structures for MbCO and deoxyMb with no apparent pathway for ligand migration through the protein matrix, dynamic protein fluctuations of side chains must be important in facilitating the ligand binding process. Near UV TRCD spectral data are fit to two exponential components with lifetimes of 110±35 μs and 1.5 ± 0.3 ms. Time-resolved absorption spectra in this wavelength region were fit to three lifetimes of 340 ± 100 ns, 830± 240μs and 2.1 ± 0.2 ms. The 830 μs and 2.1 ms processes are associated with bimolecular ligand rebinding and are consistent with lifetimes obtained from spectra in the Soret region of 200 ± 120 ns, 680 ± 80 μs and 1.8 ± 0.05 ms. The absence of the ca. 700μs component in the TRCD data indicates that bimolecular rebinding induces changes in the heme interaction with surrounding polar residues and not with aromatic residues. The 110μs TRCD transient is discussed in terms of motions of tyrosines and/or nearby aromatic groups that are involved in the early stages of ligand rebinding.

Molecular dynamics effects on protein electrostatics

Electrostatic calculations have been carried out on a number of structural conformers of tuna cytochrome c. Conformers were generated using molecular dynamics simulations with a range of solvent simulating, macroscopic dielectric formalisms, and one solvent model that explicitly included solvent water molecules. Structures generated using the lowest dielectric models were relatively tight, with side chains collapsed on the surface, while those from the higher dielectric models had more internal and external fluidity, with surface side chains exploring a fuller range of conformational space. The average structure generated with the explicitly solvated model corresponded most closely with the crystal structure. Individual pK values, overall titration curves, and electrostatic potential surfaces were calculated for average structures and structures along each simulation. Differences between structural conformers within each simulation give rise to substantial changes in calculated local electrostatic interactions, resulting in pK value fluctuations for individual sites in the protein that vary by 0.3-2.0 pK units from the calculated time average. These variations are due to the thermal side chain reorientations that produce fluctuations in charge site separations. Properties like overall titration curves and pH dependent stability are not as sensitive to side chain fluctuations within a simulation, but there are substantial effects between simulations due to marked differences in average side chain behavior. These findings underscore the importance of proper dielectric formalism in molecular dynamics simulations when used to generate alternate solution structures from a crystal structure, and suggest that conformers significantly removed from the average structure have altered electrostatic properties that may prove important in episodic protein properties such as catalysis.