Protein Conformational Equilibrium Research Articles

Protein Conformational Exchanges Modulated by the Environment of Outer Membrane Vesicles.

Protein function, in many cases, is strongly coupled to the dynamics and conformational equilibria of the protein. The environment surrounding proteins is critical for their dynamics and can dramatically affect the conformational equilibria and subsequently the activities of proteins. However, it is unclear how protein conformational equilibria are modulated by their crowded native environments. Here we reveal that outer membrane vesicle (OMV) environments modulate the conformational exchanges of Im7 protein at its local frustrated sites and shift the conformation toward its ground state. Further experiments show both macromolecular crowding and quinary interactions with the periplasmic components stabilize the ground state of Im7. Our study highlights the key role that the OMV environment plays in the protein conformational equilibria and subsequently the conformation-related protein functions. Furthermore, the long-lasting nuclear magnetic resonance measurement time of proteins within OMVs indicates that they could serve as a promising system for investigating protein structures and dynamics in situ via nuclear magnetic spectroscopy.

A De Novo Sequence Variant in Barrier-to-Autointegration Factor Is Associated with Dominant Motor Neuronopathy.

Barrier-to-autointegration factor (BAF) is an essential component of the nuclear lamina. Encoded by BANF1, this DNA binding protein contributes to the regulation of gene expression, cell cycle progression, and nuclear integrity. A rare recessive BAF variant, Ala12Thr, causes the premature aging syndrome, Néstor-Guillermo progeria syndrome (NGPS). Here, we report the first dominant pathogenic BAF variant, Gly16Arg, identified in a patient presenting with progressive neuromuscular weakness. Although disease variants carry nearby amino acid substitutions, cellular and biochemical properties are distinct. In contrast to NGPS, Gly16Arg patient fibroblasts show modest changes in nuclear lamina structure and increases in repressive marks associated with heterochromatin. Structural studies reveal that the Gly16Arg substitution introduces a salt bridge between BAF monomers, reducing the conformation ensemble available to BAF. We show that this structural change increases the double-stranded DNA binding affinity of BAF Gly16Arg. Together, our findings suggest that BAF Gly16Arg has an increased chromatin occupancy that leads to epigenetic changes and impacts nuclear functions. These observations provide a new example of how a missense mutation can change a protein conformational equilibrium to cause a dominant disease and extend our understanding of mechanisms by which BAF function impacts human health.

E-Volve: understanding the impact of mutations in SARS-CoV-2 variants spike protein on antibodies and ACE2 affinity through patterns of chemical interactions at protein interfaces.

BackgroundThe SARS-CoV-2 pandemic reverberated, posing health and social hygiene obstacles throughout the globe. Mutant lineages of the virus have concerned scientists because of convergent amino acid alterations, mainly on the viral spike protein. Studies have shown that mutants have diminished activity of neutralizing antibodies and enhanced affinity with its human cell receptor, the ACE2 protein.MethodsHence, for real-time measuring of the impacts caused by variant strains in such complexes, we implemented E-Volve, a tool designed to model a structure with a list of mutations requested by users and return analyses of the variant protein. As a proof of concept, we scrutinized the spike-antibody and spike-ACE2 complexes formed in the variants of concern, B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma), by using contact maps depicting the interactions made amid them, along with heat maps to quantify these major interactions.ResultsThe results found in this study depict the highly frequent interface changes made by the entire set of mutations, mainly conducted by N501Y and E484K. In the spike-Antibody complex, we have noticed alterations concerning electrostatic surface complementarity, breaching essential sites in the P17 and BD-368-2 antibodies. Alongside, the spike-ACE2 complex has presented new hydrophobic bonds.DiscussionMolecular dynamics simulations followed by Poisson-Boltzmann calculations corroborate the higher complementarity to the receptor and lower to the antibodies for the K417T/E484K/N501Y (Gamma) mutant compared to the wild-type strain, as pointed by E-Volve, as well as an intensification of this effect by changes at the protein conformational equilibrium in solution. A local disorder of the loop α1′/β1′, as well its possible effects on the affinity to the BD-368-2 antibody were also incorporated to the final conclusions after this analysis. Moreover, E-Volve can depict the main alterations in important biological structures, as shown in the SARS-CoV-2 complexes, marking a major step in the real-time tracking of the virus mutant lineages. E-Volve is available at http://bioinfo.dcc.ufmg.br/evolve.

All-atom adaptively biased path optimization of Src kinase conformational inactivation: Switched electrostatic network in the concerted motion of αC helix and the activation loop.

A method to optimize a conformational pathway through a space of well-chosen reduced variables is employed to advance our understanding of protein conformational equilibrium. The adaptively biased path optimization strategy utilizes unrestricted, enhanced sampling in the region of a path in the reduced-variable space to identify a broad path between two stable end-states. Application to the inactivation transition of the Src tyrosine kinase catalytic domain reveals new insight into this well studied conformational equilibrium. The mechanistic description gained from identifying the motions and structural features along the path includes details of the switched electrostatic network found to underpin the transition. The free energy barrier along the path results from rotation of a helix, αC, that is tightly correlated with motions in the activation loop (A-loop) as well as distal regions in the C-lobe. Path profiles of the reduced variables clearly demonstrate the strongly correlated motions. The exchange of electrostatic interactions among residues in the network is key to these interdependent motions. In addition, the increased resolution from an all-atom model in defining the path shows multiple components for the A-loop motion and that different parts of the A-loop contribute throughout the length of the path.

A Unique Conformational Distortion Mechanism Drives Lipocalin 2 Binding to Bacterial Siderophores.

Lcn2 is a host defense protein induced via the innate immune response to sequester iron-loaded bacterial siderophores. However, excess or prolonged elevation of Lcn2 levels can induce adverse cellular effects, including oxidative stress and inflammation. In this work, we use Hydrogen-Deuterium eXchange (HDX) and Isothermal Titration Calorimetry (ITC) to characterize the binding interaction between Lcn2 and siderophores enterobactin and 2,3-DHBA, in the presence and absence of iron. Our results indicate a rare "Type II" interaction in which binding of siderophores drives the protein conformational equilibrium toward an unfolded state. Linking our molecular model to cellular assays, we demonstrate that this "distorted binding mode" facilitates a deleterious cellular accumulation of reactive oxygen species that could represent the molecular origin of Lcn2 pathology. These results add important insights into mechanisms of Lcn2 action and have implications in Lcn2-mediated effects including inflammation.

Deciphering the Allosteric Binding Mechanism of the Human Tropomyosin Receptor Kinase A (hTrkA) Inhibitors

Access to cryptic binding pockets or allosteric sites on a kinase that present themselves when the enzyme is in a specific conformational state offers a paradigm shift in designing the next generation small molecule kinase inhibitors. The current work showcases an extensive and exhaustive array of in vitro biochemical and biophysical tools and techniques deployed along with structural biology efforts of inhibitor-bound kinase complexes to characterize and confirm the cryptic allosteric binding pocket and docking mode of the small molecule actives identified for hTrkA. Specifically, assays were designed and implemented to lock the kinase in a predominantly active or inactive conformation and the effect of the kinase inhibitor probed to understand the hTrkA binding and hTrkB selectivity. The current outcome suggests that inhibitors with a fast association rate take advantage of the inactive protein conformation and lock the kinase state by also exhibiting a slow off-rate. This in turn shifts the inactive/active state protein conformational equilibrium cycle, affecting the subsequent downstream signaling.

Differential Enzyme Flexibility Probed Using Solid-State Nanopores.

Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.

Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes

The Na+,K+-ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na+ and K+ electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state 31P and 2H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations.

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR

Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31P NMR spectra and 1H-31P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist.

Efficient implementation of constant pH molecular dynamics on modern graphics processors.

The treatment of pH sensitive ionization states for titratable residues in proteins is often omitted from molecular dynamics (MD) simulations. While static charge models can answer many questions regarding protein conformational equilibrium and protein-ligand interactions, pH-sensitive phenomena such as acid-activated chaperones and amyloidogenic protein aggregation are inaccessible to such models. Constant pH molecular dynamics (CPHMD) coupled with the Generalized Born with a Simple sWitching function (GBSW) implicit solvent model provide an accurate framework for simulating pH sensitive processes in biological systems. Although this combination has demonstrated success in predicting pKa values of protein structures, and in exploring dynamics of ionizable side-chains, its speed has been an impediment to routine application. The recent availability of low-cost graphics processing unit (GPU) chipsets with thousands of processing cores, together with the implementation of the accurate GBSW implicit solvent model on those chipsets (Arthur and Brooks, J. Comput. Chem. 2016, 37, 927), provide an opportunity to improve the speed of CPHMD and ionization modeling greatly. Here, we present a first implementation of GPU-enabled CPHMD within the CHARMM-OpenMM simulation package interface. Depending on the system size and nonbonded force cutoff parameters, we find speed increases of between one and three orders of magnitude. Additionally, the algorithm scales better with system size than the CPU-based algorithm, thus allowing for larger systems to be modeled in a cost effective manner. We anticipate that the improved performance of this methodology will open the door for broad-spread application of CPHMD in its modeling pH-mediated biological processes. © 2016 Wiley Periodicals, Inc.

The conformational response to Zn(II) and Ni(II) binding of Sporosarcina pasteurii UreG, an intrinsically disordered GTPase.

Urease is an essential Ni(II) enzyme involved in the nitrogen metabolism of bacteria, plants and fungi. Ni(II) delivery into the enzyme active site requires the presence of four accessory proteins, named UreD, UreF, UreG and UreE, acting through a complex protein network regulated by metal binding and GTP hydrolysis. The GTPase activity is catalyzed by UreG, which couples this function to a non-enzymatic role as a molecular chaperone. This moonlighting activity is reflected in a flexible fold that makes UreG the first discovered intrinsically disordered enzyme. UreG binds Ni(II) and Zn(II),which in turn modulate the interactions with other urease chaperones. The aim of this study is to understand the structural implications of metal binding to Sporosarcina pasteurii UreG (SpUreG). A combination of light scattering, calorimetry, mass spectrometry, and NMR spectroscopy revealed that SpUreG exists in monomer-dimer equilibrium (K(d)= 45 µM), sampling three distinct folding populations with different degrees of compactness. Binding of Zn(II) ions, occurring in two distinct sites (K(d1) = 3 nM, K(d2) = 0.53 µM), shifts the protein conformational landscape toward the more compact population, while maintaining the overall protein structural plasticity. Differently, binding of Ni(II) ions occurs in three binding sites (K(d1(= 14 µM; K(d2) = 270 µM; K(d3)= 160 µM), with much weaker influence on the protein conformational equilibrium. These distinct conformational responses of SpUreG to Ni(II) and Zn(II) binding suggest that selective metal binding modulates protein plasticity, possibly having an impact on the protein-protein interactions and the enzymatic activity of UreG.

Can pyrene probes be used to measure lateral pressure profiles of lipid membranes? Perspective through atomistic simulations

The lateral pressure profile of lipid bilayers has gained a lot of attention, since changes in the pressure profile have been suggested to shift the membrane protein conformational equilibrium. This relation has been mostly studied with theoretical methods, especially with molecular dynamics simulations, since established methods to measure the lateral pressure profile experimentally have not been available. The only experiments that have attempted to gauge the lateral pressure profile have been done by using di-pyrenyl-phosphatidylcholine (di-pyr-PC) probes. In these experiments, the excimer/monomer fluorescence ratio has been assumed to represent the lateral pressure in the location of the pyrene moieties. Here, we consider the validity of this assumption through atomistic molecular dynamics simulations in a DOPC (dioleoylphosphatidylcholine) membrane, which hosts di-pyr-PC probes with different acyl chain lengths. Based on the simulations, we calculate the pyrene dimerization rate and the lateral pressure at the location of the pyrenes. The dimerization rates are compared with the results of di-pyr-PC probes simulated in vacuum. The comparison indicates that the lateral pressure is not the dominant determinant of the excimer/monomer fluorescence ratio. Thus, the results do not support the usage of di-pyr-PC molecules to measure the shape of the lateral pressure profile. We yet discuss how the probes could potentially be exploited to gain qualitative insight of the changes in pressure profile when lipid composition is altered.

Insights from Free-Energy Calculations: Protein Conformational Equilibrium, Driving Forces, and Ligand-Binding Modes

Accurate free-energy calculations provide mechanistic insights into molecular recognition and conformational equilibrium. In this work, we performed free-energy calculations to study the thermodynamic properties of different states of molecular systems in their equilibrium basin, and obtained accurate absolute binding free-energy calculations for protein-ligand binding using a newly developed M2 algorithm. We used a range of Asp-Phe-Gly (DFG)-in/out p38α mitogen-activated protein kinase inhibitors as our test cases. We also focused on the flexible DFG motif, which is closely connected to kinase activation and inhibitor binding. Our calculations explain the coexistence of DFG-in and DFG-out states of the loop and reveal different components (e.g., configurational entropy and enthalpy) that stabilize the apo p38α conformations. To study novel ligand-binding modes and the key driving forces behind them, we computed the absolute binding free energies of 30 p38α inhibitors, including analogs with unavailable experimental structures. The calculations revealed multiple stable, complex conformations and changes in p38α and inhibitor conformations, as well as balance in several energetic terms and configurational entropy loss. The results provide relevant physics that can aid in designing inhibitors and understanding protein conformational equilibrium. Our approach is fast for use with proteins that contain flexible regions for structure-based drug design.

The Influence of 1-Alkanols and External Pressure on the Lateral Pressure Profiles of Lipid Bilayers

The suggestion by Robert Cantor, that drug-induced pressure changes in lipid bilayers can change the conformational equilibrium between open and closed states of membrane proteins and thereby cause anesthesia, attracted much attention lately. Here, we studied the effect of both large external pressure and of 1-alkanols of different chain lengths—some of them anesthetics, others not—on the lateral pressure profiles across dimyristoylphosphatidylcholine (DMPC) bilayers by molecular dynamics simulations. For a pure DMPC bilayer, high pressure both reduced and broadened the tension at the interface hydrophobic/hydrophilic and diminished the repulsion between the phospholipid headgroups. Whereas the effect of ethanol on the lateral pressure profile was similar to the effect of a large external pressure on a DMPC bilayer, long-chain 1-alkanols significantly amplified local maxima and minima in the lateral pressure profile. For most 1-alkanols, external pressure had moderate effects and did not reverse the changes 1-alkanols exerted on the pressure profile. Nevertheless, assuming the bent helix model as a simple geometric model for the transmembrane region of a membrane protein, protein conformational equilibria were shifted in opposite directions by addition of 1-alkanols and additional application of external pressure.

Critical Importance of Length-Scale Dependence in Implicit Modeling of Hydrophobic Interactions

The existence of length-scale dependence of hydrophobic solvation has important implications in the equilibrium of disordered, partially folded, and folded protein conformations. Neglecting this dependence, such as in popular solute surface-area based implicit solvent models with fixed surface tension coefficients, severely limits the ability to accurately model protein conformational equilibrium. We illustrate such fundamental limitations by examining the potentials of mean force of forming dimeric and trimeric nonpolar clusters and propose a new empirical model that effectively captures the context dependence of the local effective surface tension. Further optimization of the new model with other components of the implicit solvent force fields provides promise to significantly improve one's ability to simulate protein folding and conformational transitions. The existence of length-scale dependence of hydrophobic solvation has important implications in the equilibrium of disordered, partially folded, and folded protein conformations. Neglecting this dependence, such as in popular solute surface-area based implicit solvent models with fixed surface tension coefficients, severely limits the ability to accurately model protein conformational equilibrium. We illustrate such fundamental limitations by examining the potentials of mean force of forming dimeric and trimeric nonpolar clusters and propose a new empirical model that effectively captures the context dependence of the local effective surface tension. Further optimization of the new model with other components of the implicit solvent force fields provides promise to significantly improve one's ability to simulate protein folding and conformational transitions.

Kinetics of Allosteric Conformational Transition of a Macromolecule Prior to Ligand Binding: Analysis of Stopped-Flow Kinetic Experiments

Two fundamentally different mechanisms of ligand binding are commonly encountered in biological kinetics. One mechanism is a sequential multistep reaction in which the bimolecular binding step is followed by first-order steps. The other mechanism includes the conformational transition of the macromolecule, before the ligand binding, followed by the ligand binding process to one of the conformational states. In stopped-flow kinetic studies, the reaction mechanism is established by examining the behavior of relaxation times and amplitudes as a function of the reactant concentrations. A major diagnostic tool for detecting the presence of a conformational equilibrium of the macromolecule, before the ligand binding, is the decreasing value of one of the reciprocal relaxation times with the increasing [ligand]. The sequential mechanism cannot generate this behavior for any of the relaxation times. Such dependence is intuitively understood on the basis of approximate expressions for the relaxation times that can be comprehensively derived, using the characteristic equation of the coefficient matrix and polynomial theory. Generally, however, the used approximations may not be fulfilled. On the other hand, the two kinetic mechanisms can always be distinguished, using the approach based on the combined application of pseudo-first-order conditions, with respect to the ligand and the macromolecule. The two experimental conditions differ profoundly in the extent of the effect of the ligand on the protein conformational equilibrium. In a large excess of the ligand, the conformational equilibrium of the macromolecule, before the ligand binding, is strongly affected by the binding process. However, in a large excess of the macromolecule, ligand binding does not perturb the internal equilibrium of the macromolecule. As a result, the normal mode, affected by the conformational transition, is absent in the observed relaxation process. In the case of a sequential mechanism, the number of relaxation times is not altered by different pseudo-first-order conditions. Thus, the approach provides a strong diagnostic criterion for detecting the presence of the conformational transition of the macromolecule and establishing the correct mechanism. Application of this approach is illustrated for the binding of 3'-O-(N-methylantraniloyl)-5'-diphosphate to the E. coli DnaC protein.

Molecular dynamics simulations of HPr under hydrostatic pressure.

The histidine-containing protein (HPr) plays an important role in the phosphotransferase system (PTS). The deformations induced on the protein structure at high hydrostatic pressure values (4, 50, 100, 150, and 200 MPa) were previously (H. Kalbitzer, A. Görler, H. Li, P. Dubovskii, A. Hengstenberg, C. Kowolik, H. Yamada, and K. Akasaka, Protein Science 2000, Vol. 9, pp. 693-703) analyzed by NMR experiments: the nonlinear variations of the amide chemical shifts at high pressure values were supposed to arise from induced shifts in the protein conformational equilibrium. Molecular dynamics (MD) simulations are here performed, to analyze the protein internal mobility at 0.1 MPa, and to relate the nonlinear variations of chemical shifts observed at high pressure, to variations in conformational equilibrium. The global features of the protein structure are only slightly modified along the pressure. Nevertheless, the values of the Voronoi residues volumes show that the residues of alpha-helices are more compressed that those belonging to the beta-sheet. The alpha-helices are also displaying the largest internal mobility and deformation in the simulations. The nonlinearity of the 1H chemical shifts, computed from the MD simulation snapshots, is in qualitative agreement with the nonlinearity of the experimentally observed chemical shifts.

The Second Intracellular Loop of the Glycine Transporter 2 Contains Crucial Residues for Glycine Transport and Phorbol Ester-induced Regulation

Na+ and Cl(-)-coupled glycine transporters control the availability of glycine neurotransmitter in the synaptic cleft of inhibitory glycinergic pathways. In this report, we have investigated the involvement of the second intracellular loop of the neuronal glycine transporter 2 (GLYT2) on the protein conformational equilibrium and the regulation by 4alpha-phorbol 12 myristate 13-acetate (PMA). By substituting several charged (Lys-415, Lys-418, and Lys-422) and polar (Thr-419 and Ser-420) residues for different amino acids and monitoring plasma membrane expression and kinetic behavior, we found that residue Lys-422 is crucial for glycine transport. The introduction of a negative charge in 422, and to a lower extent in neighboring N-terminal residues, dramatically increases transporter voltage dependence as assessed by response to high potassium depolarizing conditions. In addition, [2-(trimethylammonium)ethyl] methanethiosulfonate accessibility revealed a conformational connection between Lys-422 and the glycine binding/permeation site. Finally, we show that the mutation of positions Thr-419, Ser-420, and mainly Lys-422 to acidic residues abolishes the PMA-induced inhibition of transport activity and the plasma membrane transporter internalization. Our results establish a new structural basis for the action of PMA on GLYT2 and suggest a complex nature of the PMA action on this glycine transporter.

A backbone-reversed all-beta polypeptide (retro-CspA) folds and assembles into amyloid nanofibres.

The backbone-reversed or 'retro', form of a model all-beta-sheet protein, Escherichia coli CspA, was produced from a synthetic gene in E.coli in fusion with an N-terminal affinity tag. Following purification under denaturing conditions and dialysis-based removal of urea, the protein was found to fold into a soluble, poorly structured multimer. Upon concentration, this state readily transformed into amyloid nanofibres. Congo Red-binding amorphous forms were also observed. Since a beta-sheet-forming sequence is expected to retain high beta-sheet-forming propensity even after backbone reversal and given the fact that folding of retro-CspA occurs only to a poorly structured form, we conclude that the increase effected in protein concentration may be responsible for the formation of intermolecular beta-sheets, facilitating the bleeding away of the protein's conformational equilibrium into aggregates that generate well-formed fibres. Since every molecule in these fibres contains a peptide tag for binding Ni(2+), the fibres may provide a template for deposition of nickel to generate novel materials.

The E. coli replication factor DnaC protein exists in two conformations with different nucleotide binding capabilities. I. Determination of the binding mechanism using ATP and ADP fluorescent analogues.

The kinetic mechanism of binding of ATP and ADP fluorescent analogues to the E. coli replicative factor DnaC protein has been studied using the fluorescence stopped-flow technique. The experiments have been performed under pseudo-first-order conditions with respect to the nucleotide cofactor or the DnaC concentration. Three relaxation processes are observed at a large excess of the nucleotide, while only two relaxation processes are detected in the excess of the protein. Such behavior of the kinetic system is a diagnostic indication of the presence of the protein conformational equilibrium prior to the ligand binding. The obtained data indicate that the minimum mechanism that describes the observed kinetics includes the conformational transition of the DnaC protein, prior to nucleotide binding, followed by the two-step, sequential association of the cofactor to only one of the protein conformations, as defined by In the examined solution conditions, the conformation of the DnaC protein is shifted toward the state (DnaC)(2) that binds the nucleotide. The lack of any cofactor binding to the (DnaC)(1) state points to the existence of a stringent locking mechanism of the nucleotide binding-site in the protein. Binding of ATP and ADP analogues obeys the same mechanism, with similar rate constants, indicating that ATP and ADP analogues bind to the same protein conformation. The (C)(1) intermediate dominates the distribution of the DnaC protein population in the presence of cofactors. The formation of (C)(1) is accompanied by a low nucleotide fluorescence increase, indicating a hydrophilic environment around the ribose of bound cofactors. Transition to (C)(2) places the ribose region in a highly hydrophobic environment with relative molar fluorescence intensity approximately 8-fold higher than that of the free cofactor. The significance of these results for the functioning of the DnaC protein is discussed.