Interdependence of conformational variables in double-helical DNA.

DNA exhibits conformational polymorphism, with the details depending on the sequence and its environment. To understand the mechanisms of conformational polymorphism and these transitions, we examine the interrelationships among the various conformational variables of DNA. In particular, we examine the stress-strain relation among conformational variables, describing base-pair morphology and their effects on the backbone conformation. For the calculation of base pairs, we use the method previously developed to calculate averages over conformational variables of DNA. Here we apply this method to calculate the Boltzmann averages of conformational variables for fixed values of one particular conformational variable, which reflects the strain in the structure responding to a particular driving stress. This averaging over all but one driving variable smooths the usual rough energy surface to permit observation of the effects of one conformational variable at a time. The stress-strain analyses of conformational variables of base pair slide, twist, and roll, which exhibit characteristic changes during the conformational transition of DNA, have shown that the conformational changes of base pairs are strongly correlated with one another. Furthermore, the stress-strain relations are not symmetrical with respect to these variables, i.e., the response of one coordinate to another is different from the reverse direction. We also examine the effect of conformational changes in base-pair variables on the sugar-backbone conformation by using the minimization method we developed. The conformational changes of base pairs affect the sugar pucker and other dihedral angles of the backbone of DNA, but each variable affects the sugar-backbone differently. In particular, twist is found to have the most influence in affecting the sugar pucker and backbone conformation. These calculated conformational changes in base pairs and backbone segments are consistent with experimental observations and serve to validate the calculation method.     

Dynamics of electron-conformational transitions in proteins and physical mechanisms of biomacromolecule function]

The proteins can be considered as a microheterogeneous structured media possessing memory and feedback properties. The conformational energy surface depends on the chemical states of protein groups. Conformational motions are local diffusion with relaxation times much longer than vibrational relaxation times in condensed media. Owing to the hierarchy of relaxation times chemical reaction rates depend on conformation parametrically. Regulation of functional activity by conformational mobility is accomplished via transmission of information in the form of changes in the distribution functions of separate groups along the conformational substates. The interpretation of drastic effects on conformational mobility needs super-stochastic approaches. A possible mechanism of sharp conformational change are discussed in terms of the catastrophe theory.     

Conformational ensemble of a multidomain protein explored by Gd3+ electron paramagnetic resonance

Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.     

Protein adsorption onto nanoparticles induces conformational changes: Particle size dependency,kinetics, and mechanisms

The use of nanomaterials in bioapplications demands a detailed understanding of protein–nanoparticle interactions. Proteins can undergo conformational changes while adsorbing onto nanoparticles, but studies on the impact of particle size on conformational changes are scarce. We have shown that conformational changes happening upon adsorption of myoglobin and BSA are dependent on the size of the nanoparticle they are adsorbing to. Out of eight initially investigated model proteins, two (BSA and myoglobin) showed conformational changes, and in both cases this conformational change was dependent on the size of the nanoparticle. Nanoparticle sizes ranged from 30 to 1000 nm and, in contrast to previous studies, we attempted to use a continuous progression of sizes in the range found in live viruses, which is an interesting size of nanoparticles for the potential use as drug delivery vehicles. Conformational changes were only visible for particles of 200 nm and bigger. Using an optimized circular dichroism protocol allowed us to follow this conformational change with regard to the nanoparticle size and, thanks to the excellent temporal resolution also in time. We uncovered significant differences between the unfolding kinetics of myoglobin and BSA. In this study, we also evaluated the plausibility of commonly used explanations for the phenomenon of nanoparticle size‐dependent conformational change. Currently proposed mechanisms are mostly based on studies done with relatively small particles, and fall short in explaining the behavior seen in our studies.     

Conformational changes in antigen-stimulated lymphocytes detected with 1-anilino-8-naphthalene sulphonate

The hydrophobic fluorescent probe, 1-anilino-8-naphthalene sulphonate (ANS), has been used to study conformational changes of mouse antigen-stimulated lymphocytes in vivo. Studies revealed that early conformational changes appear in Bovine Serum Albumin-- or Sheep Erythrocytes-- stimulated splenic and thymic lymphocytes. These conformational changes are detected by fluorescence intensity changes, when ANS is bound to lymphocytes. The kinetic studies further indicate that the course of conformational changes may vary considerably depending on antigens.     

Conformational selection or induced fit? A critical appraisal of the kinetic mechanism

For almost five decades, two competing mechanisms of ligand recognition, conformational selection and induced fit, have dominated our interpretation of ligand binding in biological macromolecules. When binding-dissociation events are fast compared to conformational transitions, the rate of approach to equilibrium, k(obs), becomes diagnostic of conformational selection or induced fit based on whether it decreases or increases, respectively, with the ligand concentration, [L]. However, this simple conclusion based on the rapid equilibrium approximation is not valid in general. Here we show that conformational selection is associated with a rich repertoire of kinetic properties, with k(obs) decreasing or increasing with [L] depending on the relative magnitude of the rate of ligand dissociation, k(off), and the rate of conformational isomerization, k(r). We prove that, even for the simplest two-step mechanism of ligand binding, a decrease in k(obs) with [L] is unequivocal evidence of conformational selection, but an increase in k(obs) with [L] is not unequivocal evidence of induced fit. Ligand binding to glucokinase, thrombin, and its precursor prethrombin-2 are used as relevant examples. We conclude that conformational selection as a mechanism for a ligand binding to its target may be far more common than currently believed.     

An application of experimental design using mutually orthogonal Latin squares in conformational studies of peptides

We address the question-can we use experimental design methods to investigate peptide conformation and identify conformational parameters that may contribute more significantly to the potential energy than others? We used mutually orthogonal Latin square design to sample the conformational space of peptides and analysed the samples using analysis of variance. We examined the equality of the effect of the torsion angles on the conformational potential energy. The results showed that different torsion angles contributed differently to the conformational energy. We are able to identify those parameters that may have to be more carefully considered in conformational studies of peptides.     

Non-Watson-Crick structures in oligodeoxynucleotides: self-association of d(TpCpGpA) stabilized at acidic pH

The 1H NMR spectrum of the tetradeoxynucleotide d(TpCpGpA) was examined as a function of temperature, pH, and concentration. At pH 7 and above the solution conformation for this oligodeoxynucleotide appears to be a mixture of random coil and Watson-Crick duplex. At 25 degrees C, a pH titration of d(TpCpGpA) shows that distinct conformational changes occur as the pH is lowered below 7.0. These conformational changes are reversible upon readjusting the pH to neutrality, indicating the presence of a pH-dependent set of conformational equilibria. At 25 degrees C, the various conformational states in the mixture are in rapid exchange on the NMR time scale. Examination of the titration curve shows the presence of distinct conformational states at pH greater than 7, and between pH 4 and pH 5. At pH less than 4, a third conformational state is present. When the pH titration is repeated at 5 degrees C, the conformational equilibria are in slow exchange on the NMR time scale; distinct signals from each conformational state are observable. The stable conformational state present between pH 4 and pH 5 represents an ordered conformation of d(TpCpGpA) which dissociates to a less ordered structure upon raising the temperature. This ordered conformation does not result from an intramolecular rearrangement, as is shown by by spectra obtained by varying oligodeoxynucleotide concentration at constant pH. The ordered conformation differs from the Watson-Crick helix, as is shown from nuclear Overhauser enhancement experiments, as well as chemical shift data. An ordered conformation for d(TpCpGpA) was previously reported [Reid, D. G., Salisbury, S. A., Brown, T., & Williams, D. H. (1985) Biochemistry 24, 4325-4332].(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of neural memory, loading factor, its regulation and optimization]

Recording and retrieving functions of the neural memory are simulated as a control of local conformational processes in neural synaptic fields. The localization of conformational changes is related to the afferent temporal-spatial pulse pattern flow, the microstructure of connections and a plurality of temporal delays in synaptic fields and afferent pathways. The loci of conformations are described by sets of afferent addresses named address domains. Being superimposed on each other, address domains form a multilayer covering of the address space of the neuron or the ensemble. The superposition factor determines the dissemination of the conformational process, and the fuzzing of memory, and its accuracy and reliability. The engram is formed as detects in the packing of the address space and hence can be retrieved in inverse form. The accuracy of the retrieved information depends on the threshold level of conformational transitions, the distribution of conformational changes in synaptic fields of the neuronal population, and the memory loading factor. The latter is represented in the model by a slow potential. It reflects total conformational changes and displaces the membrane potential to monostable conformational regimes, by governing the exit from the recording regime, the potentiation of the neurone, and the readiness to reproduction. A relative amplitude of the slow potential and the coefficient of postconformational modification of ionic conductivity, which provides maximum reliability, accuracy, and capacity of memory, are calculated.     

Additivity and non-additivity effects in the conformational changes of 1,2-ethanediol diformatein vacuo and in solution.Ab-initio and molecular mechanics descriptions

The performances of MM2 andab-initio SCF STO-3G calculations to describe conformational changes in (CH₂OCHO)₂ in vacuo and in solution are examined. We present and justify a simple procedure to add solvent effects to the MM2 conformational energies. The analysis is focused on the detection of non-additive effects in simultaneous conformational changes. We have found that the -CH₂-CH₂-group is generally effective in decoupling simultaneous conformational changes. The non-additive effects, actually present in specific classes of conformational changes, are reduced by a solvent contribution when full SCF calculations are employed, and emphasized when MM2 + solvent calculations are employed. A rationale of this finding is presented, and some suggestions are made for the correction of this artifact, due to the use of rigid charges.     

Geometry-based sampling of conformational transitions in proteins

The fast and accurate prediction of protein flexibility is one of the major challenges in protein science. Enzyme activity, signal transduction, and ligand binding are dynamic processes involving essential conformational changes ranging from small side chain fluctuations to reorientations of entire domains. In the present work, we describe a reimplementation of the CONCOORD approach, termed tCONCOORD, which allows a computationally efficient sampling of conformational transitions of a protein based on geometrical considerations. Moreover, it allows for the extraction of the essential degrees of freedom, which, in general, are the biologically relevant ones. The method rests on a reliable estimate of the stability of interactions observed in a starting structure, in particular those interactions that change during a conformational transition. Applications to adenylate kinase, calmodulin, aldose reductase, T4-lysozyme, staphylococcal nuclease, and ubiquitin show that experimentally known conformational transitions are faithfully predicted.     

Conformational flexibility of two RNA trimers explored by computational tools and database search

Two RNA sequences, AAA and AUG, were studied by the conformational search program CICADA and by molecular dynamics (MD) in the framework of the AMBER force field, and also via thorough PDB database search. CICADA was used to provide detailed information about conformers and conformational interconversions on the energy surfaces of the above molecules. Several conformational families were found for both sequences. Analysis of the results shows differences, especially between the energy of the single families, and also in flexibility and concerted conformational movement. Therefore, several MD trajectories (altogether 16 ns) were run to obtain more details about both the stability of conformers belonging to different conformational families and about the dynamics of the two systems. Results show that the trajectories strongly depend on the starting structure. When the MD start from the global minimum found by CICADA, they provide a stable run, while MD starting from another conformational family generates a trajectory where several different conformational families are visited. The results obtained by theoretical methods are compared with the thorough database search data. It is concluded that all except for the highest energy conformational families found in theoretical result also appear in experimental data. Registry numbers: adenylyl-(3' --> 5')-adenylyl-(3' --> 5')-adenosine [917-44-2] adenylyl-(3' --> 5')-uridylyl-(3' --> 5')-guanosine [3494-35-7].     

Comparison of conformational features of staphylococcal nuclease in ternary complexes with pdTp, pdGp, and nitrophenyl-pdTp.

The conformations of wild-type staphylococcal nuclease (SNase) in the ternary complexes with thymidine 3',5'-bisphosphate (pdTp), 2'-deoxyguanine 3',5'-bisphosphate (pdGp), and thymidine 3'-phosphate 5'-(p-nitrophenylphosphate) (NpdTp) with Ca2+ were examined by two-dimensional NMR NOESY and ROESY experiments. The results of these experiments indicate that the conformational features of the SNase are quite similar in the three ternary complexes. This suggests that the conformational features of SNase, in these ternary complexes, are not strongly dependent on whether the 5'-phosphate is a mono- or diester. This is in contrast to our prior studies on substitutions of active site charged amino acids which indicated that the conformational features of SNase in the ternary complex are quite sensitive to substitutions for active site charged amino acids (Hibler et al., 1987; Wilde et al., 1988; Pourmotabbed et al., 1990). The similarity of the SNase conformational features in the ternary complexes with pdTp and pdGp indicates that the features of the nucleotide bound at the active site are not strong determinants of the enzyme conformation in the ternary complexes. These conclusions are in general agreement with the results on pdApdT ternary complexes with SNase which suggested that it is the conformational features of the bound nucleic acid which determine the differences in catalysis observed for SNase with different substrates (Weber et al., 1991), more so than the conformational features of the enzyme.     

Conformational selection in protein binding and function

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational‐selection and induced‐change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational‐selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced‐change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on‐ and off‐rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single‐molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational‐selection and induced‐change processes in both binding and unbinding direction.     

Hemocyanin from E. californicum encapsulated in silica gels: oxygen binding and conformational states

Cooperativity depends on the existence of equilibria among functionally distinct conformational states that are affected by homo and heterotropic effectors. In order to isolate the quaternary conformations of hemocyanin from E. californicum, the 24-meric giant protein was encapsulated in wet, nanoporous silica gels, either in the absence or presence of oxygen. The deoxy- and oxy-hemocyanin gels exhibit a p50 for oxygen of 11 and 2.5 torr, respectively, values in close agreement with those for hemocyanin in solution. The observed Hill coefficients are lower than unity, indicating a conformational heterogeneity within each locked conformational state, a finding in agreement with the assumption that at least four conformational states are required to explain the oxygen binding properties of E. californicum hemocyanin in solution.     

An analysis of side-chain conformation in proteins.

The crystal structures of a number of globular proteins are currently available. An analysis of the distribution of side-chains among different allowed conformations in these proteins has been carried out. The observed conformations of individual residues are discussed on the basis of well-known stereochemical criteria. The population distribution of side-chains in different allowed regions in conformational space can be explained largely on the basis of simple steric considerations. In addition to examining the conformational behaviour of individual residues, some population distributions of conformational angles of general interest involving groups of residues have also been analyzed.     

A Physical Picture of Protein Dynamics and Conformational Changes

A physical model is reviewed which explains different aspects of protein dynamics consistently. At low temperatures, the molecules are frozen in conformational substates. Their average energy is 3/2RT. Solid-state vibrations occur on a time scale of femtoseconds to nanoseconds. Above a characteristic temperature, often called the dynamical transition temperature, slow modes of motions can be observed occurring on a time scale between about 140 and 1 ns. These motions are overdamped, quasidiffusive, and involve collective motions of segments of the size of an α-helix. Molecules performing these types of motion are in the “flexible state”. This state is reached by thermal activation. It is shown that these motions are essential for conformational relaxation. Based on this picture, a new approach is proposed to understand conformational changes. It connects structural fluctuations and conformational transitions.     

Speed of Conformational Change: Comparing Explicit and Implicit Solvent Molecular Dynamics Simulations

Adequate sampling of conformation space remains challenging in atomistic simulations, especially if the solvent is treated explicitly. Implicit-solvent simulations can speed up conformational sampling significantly. We compare the speed of conformational sampling between two commonly used methods of each class: the explicit-solvent particle mesh Ewald (PME) with TIP3P water model and a popular generalized Born (GB) implicit-solvent model, as implemented in the AMBER package. We systematically investigate small (dihedral angle flips in a protein), large (nucleosome tail collapse and DNA unwrapping), and mixed (folding of a miniprotein) conformational changes, with nominal simulation times ranging from nanoseconds to microseconds depending on system size. The speedups in conformational sampling for GB relative to PME simulations, are highly system- and problem-dependent. Where the simulation temperatures for PME and GB are the same, the corresponding speedups are approximately onefold (small conformational changes), between ∼1- and ∼100-fold (large changes), and approximately sevenfold (mixed case). The effects of temperature on speedup and free-energy landscapes, which may differ substantially between the solvent models, are discussed in detail for the case of miniprotein folding. In addition to speeding up conformational sampling, due to algorithmic differences, the implicit solvent model can be computationally faster for small systems or slower for large systems, depending on the number of solute and solvent atoms. For the conformational changes considered here, the combined speedups are approximately twofold, ∼1- to 60-fold, and ∼50-fold, respectively, in the low solvent viscosity regime afforded by the implicit solvent. For all the systems studied, 1) conformational sampling speedup increases as Langevin collision frequency (effective viscosity) decreases; and 2) conformational sampling speedup is mainly due to reduction in solvent viscosity rather than possible differences in free-energy landscapes between the solvent models.     

Evolutionary Conserved Positions Define Protein Conformational Diversity

Conformational diversity of the native state plays a central role in modulating protein function. The selection paradigm sustains that different ligands shift the conformational equilibrium through their binding to highest-affinity conformers. Intramolecular vibrational dynamics associated to each conformation should guarantee conformational transitions, which due to its importance, could possibly be associated with evolutionary conserved traits. Normal mode analysis, based on a coarse-grained model of the protein, can provide the required information to explore these features. Herein, we present a novel procedure to identify key positions sustaining the conformational diversity associated to ligand binding. The method is applied to an adequate refined dataset of 188 paired protein structures in their bound and unbound forms. Firstly, normal modes most involved in the conformational change are selected according to their corresponding overlap with structural distortions introduced by ligand binding. The subspace defined by these modes is used to analyze the effect of simulated point mutations on preserving the conformational diversity of the protein. We find a negative correlation between the effects of mutations on these normal mode subspaces associated to ligand-binding and position-specific evolutionary conservations obtained from multiple sequence-structure alignments. Positions whose mutations are found to alter the most these subspaces are defined as key positions, that is, dynamically important residues that mediate the ligand-binding conformational change. These positions are shown to be evolutionary conserved, mostly buried aliphatic residues localized in regular structural regions of the protein like β-sheets and α-helix.