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PubMed2006

On the analysis of conformational dynamics in polymers with several rotational isomers.

The ability of different correlation functions to shed some light onto the conformational dynamics of an amorphous polymer has been analyzed. The study has been performed on a polyethylene model polymer, which has been simulated at decreasing temperatures towards its glass transition, via the molecular dynamics technique. Three rotational isomers are allowed by the considered torsional potential. The correlation times associated with the evaluated transition rates have shown to be Arrhenius in nature, with activation energies resulting basically from internal rotation barriers. Overall torsional autocorrelation functions have been calculated. We have observed that they are dominated by slow events. Alternatively, a set of torsional autocorrelation functions associated with every isomeric state has been evaluated. Stretched exponential fits lead to correlation times that display Vogel-Fulcher temperature dependence.

Journal Article↗
PubMed1993

Human factor VIIa and its complex with soluble tissue factor: evaluation of asymmetry and conformational dynamics by ultracentrifugation and fluorescence anisotropy decay methods.

Ultracentrifugation and fluorescence anisotropy decay measurements were used to evaluate the asymmetry and conformational dynamics of human blood clotting enzyme VIIa (VIIa) and the complex it forms with a soluble truncation mutant of human tissue factor (sTF) which acts as an essential cofactor for VIIa. Sedimentation velocity experiments showed that both VIIa and the sTF.VIIa complex are highly asymmetric. In each case, the friction ratio f/fsphere, is consistent with a family of general elliposids ranging from prolate to oblate. Fluorescence anisotropy decay experiments were used to limit the family of elliposids which can describe the hydrodynamic behavior of VIIa and sTF.VIIa. For both VIIa and the sTF.VIIa complex, the oblate ellipsoid of revolution was eliminated. In addition, the fluorescence anisotropy decay data clearly show that upon binding sTF.VIIa loses a segmental motion involving a domain containing the active site of the enzyme. This suggests that sTF causes a stabilization of a limited range of VIIa conformations. This stabilization may be important for proper recognition of the TF.VIIa substrate, factor X.

Chemical Phenomena↗
PubMed2006

Shedding light on biomolecule conformational dynamics using fluorescence measurements of trapped ions.

Biomolecule conformational change has been widely investigated in solution using several methods; however, much less experimental data about structural changes are available for completely isolated, gas-phase biomolecules. Studies of conformational change in unsolvated biomolecules are required to complement the interpretation of mass spectrometry measurements and in addition, can provide a means to directly test theoretical simulations of biomolecule structure and dynamics independent of a simulated solvent. In this Feature Article, we review our recent introduction of a fluorescence-based method for probing local conformational dynamics in unsolvated biomolecules through interactions of an attached dye with tryptophan (Trp) residues and fields originating on charge sites. Dye-derivatized biomolecule ions are formed by electrospray ionization and are trapped in a variable-temperature quadrupole ion trap in which they are irradiated with either continuous or short pulse lasers to excite fluorescence. Fluorescence is measured as a function of temperature for different charge states. Optical measurements of the dye fluorescence include average intensity changes, changes in the emission spectrum, and time-resolved measurements of the fluorescence decay. These measurements have been applied to the miniprotein, Trp-cage, polyproline peptides and to a beta-hairpin-forming peptide, and the results are presented as examples of the broad applicability and utility of these methods. Model fits to Trp-cage fluorescence data measured as a function of temperature provide quantitative information on the thermodynamics of conformational changes, which are reproduced well by molecular dynamics. Time-resolved measurements of the fluorescence decays of Trp-cage and small polyproline peptides definitively demonstrate the occurrence of fluorescence quenching by the amino acid Trp in unsolvated biomolecules.

Fluorescence↗
PubMed2001

Essential dynamics of reversible peptide folding: memory-free conformational dynamics governed by internal hydrogen bonds.

A principal component analysis has been applied on equilibrium simulations of a beta-heptapeptide that shows reversible folding in a methanol solution. The analysis shows that the configurational space contains only three dense sub-states. These states of relatively low free energy correspond to the "native" left-handed helix, a partly helical intermediate, and a hairpin-like structure. The collection of unfolded conformations form a relatively diffuse cloud with little substructure. Internal hydrogen-bonding energies were found to correlate well with the degree of folding. The native helical structure folds from the N terminus; the transition from the major folding intermediate to the native helical structure involves the formation of the two most C-terminal backbone hydrogen bonds. A four-state Markov model was found to describe transition frequencies between the conformational states within error limits, indicating that memory-effects are negligible beyond the nanosecond time-scale. The dominant native state fluctuations were found to be very similar to unfolding motions, suggesting that unfolding pathways can be inferred from fluctuations in the native state. The low-dimensional essential subspace, describing 69% of the collective atomic fluctuations, was found to converge at time-scales of the order of one nanosecond at all temperatures investigated, whereas folding/unfolding takes place at significantly longer time-scales, even above the melting temperature.

Computer Simulation↗
PubMed2003

Thermodynamic and kinetic analysis of a peptide-class I MHC interaction highlights the noncovalent nature and conformational dynamics of the class I heterotrimer.

The class I major histocompatibility (MHC) molecule is a heterotrimer composed of a heavy chain, the small subunit beta(2)-microglobulin (beta(2)m), and a peptide. Fluorescence anisotropy has been used to assay the interaction of a labeled peptide with a recombinant, soluble form of the class I MHC HLA-A2. Consistent with earlier work, peptide binding is shown to be a two-step process limited by a conformational rearrangement in the heavy chain/beta(2)m heterodimer. However, we identify two pathways for peptide dissociation from the heterotrimer: (1) initial peptide dissociation leaving a heavy chain/beta(2)m heterodimer and (2) initial dissociation of beta(2)m, followed by peptide dissociation from the heavy chain. Eyring analyses of rate constants measured as a function of temperature permit for the first time a complete thermodynamic characterization of peptide binding. We find that in this case peptide binding is mostly entropically driven, likely reflecting the hydrophobic character of the peptide binding groove and the peptide anchor residues. Thermodynamic and kinetic analyses of peptide-MHC interactions as performed here may be of practical use in the engineering of peptides with desired binding properties and will aid in the interpretation of the effects of MHC and peptide substitutions on peptide binding and T cell reactivity. Finally, our data suggest a role for beta(2)m in dampening conformational dynamics in the heavy chain. Remaining conformational variability in the heavy chain once beta(2)m has bound may be a mechanism to promote promiscuity in peptide binding.

Amino Acid Sequence↗
PubMed2001

Conformational dynamics of an alanine dipeptide analog: an ab initio molecular dynamics study.

An ab initio molecular dynamics (MD) simulation technique employing the Born-Oppenheimer approach in the framework of a Gaussian implementation of Kohn-Sham density functional theory is used to study the gas-phase conformational dynamics of an alanine dipeptide analog. It is found that conformational transformation between C5 and C7(eq) occurs on the picosecond time scale. Classical MD simulations using most of the popular force fields do not yield a transition even after nanoseconds. An analysis is given of the difference, for this small gas-phase system, between ab initio MD and traditional MD simulation using force fields.

Alanine↗
PubMed2002

2-Arylindene metallocenes: conformationally dynamic catalysts to control the structure and properties of polypropylenes.

Polymerization of propylene with catalysts derived from unbridged 2-arylindene metallocenes leads to elastomeric, low-tacticity polypropylenes. The polymerization behavior of these conformationally dynamic catalyst systems is interpreted in terms of a multistate mechanism where the catalyst enchains the monomer from both stereoselective and nonstereoselective conformations during the lifetime of a single polymer chain. The structure and properties of the polypropylenes depend sensitively on the metallocene and the polymerization conditions (temperature, pressure, monomer concentration); samples of low to intermediate isotacticity ([mmmm] = 20-40%) are elastomeric, with low densities and broad melting ranges that extend to 150 degrees C. Samples of higher tacticity ([mmmm] = 40-70%) behave as softened thermoplastics.

Journal Article↗
PubMed1991

Insight into the conformational dynamics of specific regions of porcine pancreatic phospholipase A2 from a time-resolved fluorescence study of a genetically inserted single tryptophan residue.

The effects of Ca2+ and substrate analogue binding on the conformational dynamics of porcine pancreas phospholipase A2 (PLA2) in different regions was explored by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) of the wild-type protein (W3), in the alpha-helix A, was replaced by a phenylalanine residue (W3F), whereafter Trp was substituted either for leucine-31 (W31), located in the calcium binding loop, or for phenylalanine-94 (W94), located at the "back side" of the enzyme. Furthermore, mutants lacking the 62-66 sequence were constructed with the Trp at position 3 (delta W3) or 31 (delta W31). The total fluorescence intensity decays of Trp in each protein, in the protein-calcium and the protein-calcium-substrate analogue complexes, analyzed by the maximum entropy method (MEM) can be interpreted as distributions of separated lifetime classes. In the case of the W94 mutant, a major short-lived excited-state population (tau approximately 50 ps) is observed, probably deactivated by the interaction with two proximate disulfide bridges via a radiationless process. For the four other mutants, the respective barycenters of the four lifetime classes display comparable values, but the amplitude distributions are different for Trp-3 and Trp-31. The rotational mobility of the Trp residue varies along the peptide chain. Trp-3 experiences only a fast hindered motion. Trp-31 is sensitive to an additional local flexibility that is absent in the N-terminal part of the protein. The largest wobbling angle is observed at position 94. No effect of calcium binding occurs on the lifetime distribution of the Trp-3 and Trp-94 residues. Their mobilities are not affected. In contrast, calcium binding displays a strong influence on the excited-state population distribution of Trp-31. A major population decaying with the longest lifetime is selected in the W31 protein and contributes to approximately 50% of the decay. The local flexibility and the amplitude of motion of Trp-31 is wider in the protein-calcium complex than in the unliganded protein. Binding of the monomeric substrate analogue n-dodecylphosphocholine (C12PN) in the presence of calcium slightly affects the Trp-3 excited-state population distribution and its mobility. Trp-31 is more sensitive to this binding. In particular, a more restricted rotation of the Trp-31 residue and a decrease of the peptide local flexibility as protein-calcium complexes are observed in both the W31 and delta W31 mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1980

A conductance stopped-flow study of polyelectrolyte systems. Dynamic conformational change of synthetic macroions and their complexation with neutral polymers.

Two dynamic phenomena of polyelectrolyte systems, namely the rapid conformational changes of weak polyelectiolytes (polyacrylic acid (HPAA) and polyethylenimine, induced by pH-jump) and the complexation of HPAA with polyvinylpyrrolidone are first investigated by means of the conductance stopped-flow (kappa-SF) technique. The pH-induced relaxations observed by mixing an aqueous solution of HPAA or polyethylenimine with water containing a tiny amount of alkali or acid, are safely ascribed to the conformational transition of the macroions between the compact "coil" and the stretched "rod" forms. The relaxation times are large at high degrees of neutralization and for "rod" conformation. Tentative values for the thermodynamic parameters of the dynamic conformational changes are obtained and discussed. The reaction rates of the polymer complex formation and dissociation of HPAA with polyvinylpyrrolidone are also determined using the kappa-SF method. The association constant and the rates of formation and dissociation are 1.84 x 10(3) M(-1), 9.4 x 10(4) M(-1) s(-1) and 51 s(-1) at 10 degrees C, respectively. We then derive the thermodynamic parameters from the above results.

Journal Article↗
PubMed2003

Conformational dynamics of partially denatured myoglobin studied by time-resolved electrospray mass spectrometry with online hydrogen-deuterium exchange.

This study demonstrates the use of electrospray mass spectrometry in conjunction with rapid online mixing ("time-resolved" ESI-MS) for monitoring protein conformational dynamics under equilibrium conditions. The hydrogen/deuterium exchange (HDX) kinetics of mildly denatured myoglobin (Mb) at pD 9.3, in the presence of 27% acetonitrile, were studied with millisecond time resolution. Analytical ultracentrifugation indicates that the average protein compactness under these solvent conditions is similar to that of native holomyoglobin (hMb). The mass spectrum shows protein ions in a wide array of charge and heme binding states, indicating the presence of multiple coexisting conformations. The experimental approach used allows the HDX kinetics of all of these species to be monitored separately. A combination of EX1 and EX2 behavior was observed for hMb ions in charge states 7+ to 9+, which predominantly represent nativelike hMb in solution. The EX1 kinetics are biphasic, indicating the presence of two protein populations that undergo conformational opening events with different rate constants. The EX2 kinetics observed for nativelike hMb are biphasic as well. All other charge and heme binding states represent non-native protein conformations that are involved in rapid interconversion processes, thus leading to monoexponential EX2 kinetics with a common rate constant. Burst phase labeling for these non-native proteins occurs at 125 sites. In contrast, the nativelike protein conformation shows burst phase labeling only for 88 sites. A kinetic model is developed which is based on the assumption of three distinct (un)folding units in Mb. The model implies that the free energy landscape of the protein exhibits a major barrier. The crossing of this barrier is most likely associated with slow, cooperative opening/closing events of the heme binding pocket. Rapid conformational fluctuations on either side of the barrier give rise to the observed EX2 kinetics. Simulated HDX kinetics based on this model are in excellent agreement with the experimental data.

Animals↗
PubMed1989

Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements.

Fluorescence energy transfer is widely used for determination of intramolecular distances in macromolecules. The time dependence of the rate of energy transfer is a function of the donor/acceptor distance distribution and fluctuations between the various conformations which may occur during the lifetime of the excited state. Previous attempts to recover both distance distributions and segmental diffusion from time-resolved experiments have been unsuccessful due to the extreme correlation between fitting parameters. A method has been developed, based on global analysis of both donor and acceptor fluorescence decay curves, which overcomes this extreme cross-correlation and allows the parameters of the equilibrium distance distributions and intramolecular diffusion constants to be recovered with high statistical significance and accuracy. Simulation studies of typical intramolecular energy transfer experiments reveal that both static and dynamic conformational distribution information can thus be obtained at a single temperature and viscosity.

Biopolymers↗
PubMed1986

Conformational dynamics of two histidine-binding proteins of Salmonella typhimurium.

The Salmonella typhimurium periplasmic histidine-binding J-protein is one of four proteins encoded by the histidine transport operon. Mutant J-protein hisJ5625 binds L-histidine, but does not transport it. The tertiary structure and conformational dynamics of native and mutant J-protein have been compared using steady state fluorescence, fluorescence polarization, and fluorescence energy transfer measurements. The two proteins have different three-dimensional structures and exhibit different responses to histidine binding. Ligand-induced conformational changes were demonstrated in both J-proteins using fluorescence energy transfer (distant reporter method) between the single tryptophan residue per mole of protein and a fluorescein-labeled methionine residue. However, the conformational change of the mutant protein is qualitatively and quantitatively different from that of the wild-type protein. Moreover, the microenvironment of the tryptophan and its distance from the labeled methionine (44A for the wild type, 60A for the mutant J-protein) are different in the two proteins. In conclusion, these results indicate that the specific conformational change induced in the wild type J-protein is a necessary requirement for the transport of L-histidine.

Carrier Proteins↗
PubMed2005

Conformational dynamics of M2 helices in KirBac channels: helix flexibility in relation to gating via molecular dynamics simulations.

KirBac1.1 and 3.1 are bacterial homologues of mammalian inward rectifier K channels. We have performed extended molecular dynamics simulations (five simulations, each of >20 ns duration) of the transmembrane domain of KirBac in two membrane environments, a palmitoyl oleoyl phosphatidylcholine bilayer and an octane slab. Analysis of these simulations has focused on the conformational dynamics of the pore-lining M2 helices, which form the cytoplasmic hydrophobic gate of the channel. Principal components analysis reveals bending of M2, with a molecular hinge at the conserved glycine (Gly134 in KirBac1.1, Gly120 in KirBac3.1). More detailed analysis reveals a dimer-of-dimers type motion. The first two eigenvectors describing the motions of M2 correspond to helix kink and swivel motions. The conformational flexibility of M2 seen in these simulations correlates with differences in M2 conformation between that seen in the X-ray structures of closed channels (KcsA and KirBac) in which the helix is undistorted, and in open channels (e.g. MthK) in which the M2 helix is kinked. Thus, the simulations, albeit on a time scale substantially shorter than that required for channel gating, suggest a gating model in which the intrinsic flexibility of M2 about a molecular hinge is coupled to conformational transitions of an intracellular 'gatekeeper' domain, the latter changing conformation in response to ligand binding.

Animals↗
PubMed1999

Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism.

Fluorescence resonance energy transfer and fluorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular fluorescence resonance energy transfer and fluorescence polarization anisotropy measurements of fluorescently labeled staphylococcal nuclease molecules reveal distinct patterns of fluctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular fluorescence resonance energy transfer measurements provide information about the dynamic interactions of staphylococcal nuclease with single substrate molecules. The experimental methods demonstrated here should prove generally useful in studies of protein folding and enzyme catalysis at single-molecule resolution.

Base Sequence↗
PubMed2006

Bridging conformational dynamics and function using single-molecule spectroscopy.

In a typical structure-function relation study, the primary structure of proteins or nucleic acids is changed by mutagenesis and its functional effect is measured via biochemical means. Single-molecule spectroscopy has begun to give a whole new meaning to the "structure-function relation" by measuring the real-time conformational changes of individual biological macromolecules while they are functioning. This review discusses a few recent examples: untangling internal chemistry and conformational dynamics of a ribozyme, branch migration landscape of a Holliday junction at a single-step resolution, tRNA selection and dynamics in a ribosome, repetitive shuttling and snapback of a helicase, and discrete rotation of an ATP synthase.

Adenosine Triphosphate↗
PubMed2003

Conformational dynamics of beta(2)-microglobulin analyzed by reduction and reoxidation of the disulfide bond.

Although native beta(2)-microglobulin (beta2-m), the light chain of the major histocompatibility complex class I antigen, assumes an immunoglobulin domain fold, it is also found as a major component of dialysis-related amyloid fibrils. In the amyloid fibrils, the conformation of beta2-m is considered to be largely different from that of the native state, and a monomeric denatured form is likely to be a precursor to the amyloid fibril. To obtain insight into the conformational dynamics of beta2-m leading to the formation of amyloid fibrils, we studied the reduction and reoxidation of the disulfide bond by reduced and oxidized dithiothreitol, respectively, and the effects on the reduction of the chaperonin GroEL, a model protein that might destabilize the native state of beta2-m. We show that beta2-m occasionally unfolds into a denatured form even under physiological conditions and that this transition is promoted upon interaction with GroEL. The results imply that in vivo interactions of beta2-m with other proteins or membrane components could destabilize its native structure, thus stabilizing the amyloid precursor.

Amyloid↗
PubMed2002

Conformational dynamics of helix S6 from Shaker potassium channel: simulation studies.

Prolines in transmembrane (TM) alpha-helices are believed to play an important structural and/or functional role in membrane proteins. At a structural level a proline residue distorts alpha-helical structure due to the loss of at least one stabilizing backbone hydrogen bond, and introduces flexibility in the helix that may result in substantial kink and swivel motions about the effective "hinge." At a functional level, for example in Kv channels, it is believed that proline-induced molecular hinges may have a direct role in gating, i.e., the conformational change linked to opening/closing the channel to movement of ions. In this article we study the conformational dynamics of the S6 TM helix from of the Kv channel Shaker, which possesses the motif PVP--a motif that is conserved in Kv channels. We perform multiple molecular dynamics simulations of single S6 helices in a membrane-mimetic environment in order to effectively map the kink-swivel conformational space of the protein, exploiting the ability of multiple simulations to achieve greater sampling. We show that the presence of proline locally perturbs the helix, disrupting local dihedral angles and producing local twist and unwinding in the region of the hinge--an effect that is relaxed with distance from the PVP motif. We furthermore show that motions about the hinge are highly anisotropic, reflecting a preferred region of kink-swivel conformation space that may have implications for the gating process.

Models, Molecular↗