Role Of Conformational Changes Research Articles

Reordering Hydrogen Bonds In The Protein Backbone In Hamiltonian Exchange MD Enhances Sampling Of Conformational Changes

Molecular simulation enables detailed insight into the mechanisms underlying protein conformational changes and is complementary to experiments. Studying a protein folding reaction at atomistic resolution with conventional Molecular Dynamics (MD) is unpractical due to the long time scales involved. These long time scales originate from the presence of local free energy minima from which it is not trivial to escape.The replica exchange MD (REMD) method allows for the escape of such local minima by letting multiple replicas diffuse through temperature space while maintaining the canonical distribution at the temperature of interest. Instead of switching temperatures, the replicas can also exchange (part of) their Hamiltonians. A smooth interpolation between the regular Hamiltonian and one that enables swift conformational changes of the protein (e.g. by lowering barriers) enhances sampling of the conformational space.As hydrogen bonds play an important role in conformational changes of proteins, including folding, it seems natural to bias these bonds specifically. We have developed biasing potentials that allow the breaking and formation of bonds between arbitrary hydrogen bond donors and acceptors in the protein backbone. Employing these potentials in a Hamiltonian REMD scheme we aim to reorder hydrogen bonds in the protein backbone, and thus speed up the conformational sampling.For a simple beta-heptapeptide test system, we show that our hydrogen bond switching scheme is four times more efficient in sampling the conformational space of the peptide as conventional temperature REMD, while it also has the advantage of not having to know the stable states in advance.Finally, we discuss the slow convergence for larger systems, which is often observed in REMD or Hamiltonian exchange. This is caused by replicas having a preference for specific biasing potentials.

Diversification of West Nile virus in a subtropical region

BackgroundWest Nile virus (WNV) has spread across North, Central, and South America since its introduction in 1999. At the start of this spread, Florida was considered a potentially important area with regards to transmission due to its geographic, climatological, and demographic conditions. Curiously, the anticipated high levels of transmission or disease outbreaks have not been observed. As other studies have predicted that the lack of intense WNV transmission is not due to vector incompetence, we sought to evaluate the role of viral strain diversity in WNV transmission in Florida. Therefore, a phylogentic analysis was carried out on several isolates collected from three distinct locations in Florida.ResultsContrasting with a positive control collected in Indian River County, Florida during 2003 that contains the original NY99 genotype with valanine at amino acid 159 of the envelope region, all of the isolates collected in 2005 contain the WN02 genotype composed of a substation with alanine at that position indicating the window of introduction of the WN02 genotype occurred between 2003 and 2005. From the eight isolates collected in Duval, Indian River, and Manatee Counties; there is also a silent nucleotide substitution that differentiates the isolates collected on the Atlantic side of the state compared to the isolate collected on the Gulf side, which groups closer to isolates from other locations near the Gulf.ConclusionAs a whole, the Florida isolates contained numerous variable nucleotide and amino acid sites from the reference sequences, as well as each other; indicating greater nucleotide diversity within the Florida 2005 isolates than within other regions. Finally, a series of three amino acid substitutions surrounding a set of histidines located in the envelope coding region that hypothesized to play a role in conformational changes was found in the isolate collected in Indian River County, perhaps changing the antigenicity of the homodimer. Taken together, these findings expand our understanding of the temporal and spatial compartmentalization of West Nile virus subtypes within North America.

Kinetic Analysis of Correct Nucleotide Insertion by a Y-family DNA Polymerase Reveals Conformational Changes Both Prior to and following Phosphodiester Bond Formation as Detected by Tryptophan Fluorescence

The Sulfolobus solfataricus Y-family DNA polymerase Dpo4 is a model for translesion replication and has been used in the analysis of individual steps involved in catalysis. The role of conformational changes has not been clear. Introduction of Trp residues into the Trp-devoid wild-type protein provided fluorescence probes of these events, particularly in the case of mutants T239W and N188W. With both mutants, a rapid increase in Trp fluorescence was observed only in the case of normal base pairing (G:C), was saturable with respect to dCTP concentration, and occurred in the absence of phosphodiester bond formation. A subsequent decrease in the Trp fluorescence occurred when phosphodiester bond formation was permitted, and these rates were independent of the dCTP concentration. This step is relatively slow and is attributed to a conformational relaxation step occurring after pyrophosphate release, which was measured and shown to be fast in a separate experiment. The measured rate of release of DNA from Dpo4 was rapid and is not rate-limiting. Overall, the measurements provide a kinetic scheme for Dpo4 different than generally accepted for replicative polymerases or proposed for Dpo4 and other Y-family polymerases: the initial enzyme.DNA.dNTP complex undergoes a rapid (18 s(-1)), reversible (21 s(-1)) conformational change, followed by relatively rapid phosphodiester bond formation (11 s(-1)) and then fast release of pyrophosphate, followed by a rate-limiting relaxation of the active conformation (2 s(-1)) and then rapid DNA release, yielding an overall steady-state kcat of <1 s(-1).

Conformational changes in an ultrafast light-driven enzyme determine catalytic activity

The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology. Although it is now widely regarded that enzymes modulate reaction rates by means of short- and long-range protein motions, it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers. Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excited-state lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.

Role of conformational change in the C-terminus of β2-microglobulin in dialysis-related amyloidosis

Beta(2)-microglobulin (beta(2)m) has been identified as the precursor protein of dialysis-related amyloidosis (DRA), which is a serious complication for haemodialysis (HD) patients. However, mechanisms underlying beta(2)m amyloid fibril formation remains to be elucidated. We previously demonstrated, in amyloid deposits from HD patients, a conformational isoform of beta(2)m with an unfolded C-terminus. However, no direct experiments have previously been performed to address whether unfolded beta(2)m in the C-terminus may be prone to form amyloid fibrils. To evaluate roles of C-terminal amino acids in beta(2)m-induced amyloid formation, we generated six types of recombinant beta(2)m with amino acid substitutions in the C-terminal region. To investigate their conformational change and amyloidogenicity, we measured circular dichroism spectra, the fluorescence intensity of tryptophan and thioflavin-T (ThT) of the recombinant beta(2)m. To analyse morphological change of beta(2)m, we performed electron microscopy (EM) on the samples with elevated ThT fluorescence intensity. We used ultrasonication to enhance beta(2)m destabilization of the protein. Beta(2)M Trp95Leu and Arg97Ala showed conformational changes and increased their amyloidgenicity compared with beta(2)m wild-type (WT). With ultrasonication, beta(2)m Trp95Leu and Arg97Ala generated more amyloid fibrils than did beta(2)m WT even in physiological solution. EM showed that beta(2)m formed amorphous debris containing typical amyloid fibrils at 24 hours, when ThT fluorescence intensity was three-fold lower than that at six hours. Conformational changes in the C-terminus of beta(2)m may play an important role in DRA and that ultrasonication is useful for analysis of beta(2)m amyloidogenesis.

Amino Acid Residues Involved in Membrane Insertion and Pore Formation of Clostridium botulinum C2 Toxin

The actin-ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzymatic component C2I and the binding component C2II. C2II forms heptameric channels involved in translocation of the enzymatic component into the target cell. On the basis of the heptameric toxin channel, we studied functional consequences of mutagenesis of amino acid residues probably lining the lumen of the toxin channel. Substitution of glutamate-399 of C2II with alanine blocked channel formation and cytotoxicity of the holotoxin. Although cytotoxicity and rounding up of cells by C2I were completely blocked by exchange of phenylalanine-428 with alanine, the mutation increased potassium conductance caused by C2II in artificial membranes by about 2-3-fold over that of wild-type toxin. In contrast to its effects on single-channel potassium conductance in artificial membranes, the F428A mutation delayed the kinetics of pore formation in lipid vesicles and inhibited the activity of C2II in promoting (86)Rb (+) release from preloaded intact cells after pH shift of the medium. Moreover, F428A C2II exhibited delayed and diminished formation of C2II aggregates at low pH, indicating major changes of the biophysical properties of the toxin. The data indicate that phenylalanine-428 of C2II plays a major role in conformational changes occurring during pore formation of the binding component of C2II.

Substrate specificity and conformational preferences of prolyl 4‐hydroxylase

Prolyl 4‐hydroxylase (P4H) catalyzes the post‐translational hydroxylation of proline residues in procollagen strands. P4H specifically forms 4(R)‐hydroxyproline (Hyp) from proline residues in Pro–Gly sequences of polypeptides. The presence of Hyp is crucial for the folding, secretion, and stability of the collagen triple helix under physiological conditions.The current study examines the substrate recognition and specificity of P4H. We have synthesized peptide substrate analogs containing 4(R)‐fluoro‐L‐proline (Flp), 4(S)‐fluoro‐L‐proline (flp), and 4‐thio‐L‐proline (Thp). The peptide containing Flp was not found to interact with P4H, whereas the flp‐ and Thp‐containing peptides were determined to be substrates of P4H, forming products containing 4‐keto‐L‐proline and 4(R)‐sulfoxoproline. These results suggest that the cis/trans conformation of the substrate is a factor in recognition by P4H, and provide evidence for a role of conformational changes in collagen biosynthesis.This work is supported by Grant AR44276 (NIH). KLG was supported by the NIH Chemistry–Biology Interface training program.

Ligand-induced Conformational Changes and Conformational Dynamics in the Solution Structure of the Lactose Repressor Protein

We present here the results of a series of small-angle X-ray scattering studies aimed at understanding the role of conformational changes and structural flexibility in DNA binding and allosteric signaling in a bacterial transcription regulator, lactose repressor protein (LacI). Experiments were designed to detect possible conformational changes that occur when LacI binds either DNA or the inducer IPTG, or both. Our studies included the native LacI dimer of homodimers and a dimeric variant (R3), enabling us to probe conformational changes within the homodimers and distinguish them from those involving changes in the homodimer–homodimer relationships. The scattering data indicate that removal of operator DNA (oDNA) from R3 results in an unfolding and extension of the hinge helix that connects the LacI regulatory and DNA-binding domains. In contrast, only very subtle conformational changes occur in the R3 dimer–oDNA complex upon IPTG binding, indicative of small adjustments in the orientations of domains and/or subdomains within the structure. The binding of IPTG to native (tetrameric) LacI–oDNA complexes also appears to facilitate a modest change in the average homodimer–homodimer disposition. Notably, the crystal structure of the native LacI–oDNA complex differs significantly from the average solution conformation. The solution scattering data are best fit by an ensemble of structures that includes (1) ∼60% of the V-shaped dimer of homodimers observed in the crystal structure and (2) ∼40% of molecules with more “open” forms, such as those generated when the homodimers move with respect to each other about the tetramerization domain. In gene regulation, such a flexible LacI would be beneficial for the interaction of its two DNA-binding domains, positioned at the tips of the V, with the required two of three LacI operators needed for full repression.

Substrate-induced Conformational Changes and Dynamics of UDP- N-Acetylgalactosamine:Polypeptide N-Acetylgalactosaminyltransferase-2

O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide: N-acetyl-α-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.

The Chloride Dependence of the Human Organic Anion Transporter 1 (hOAT1) Is Blunted by Mutation of a Single Amino Acid

Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.

The Crystal Structure and Mutational Binding Analysis of the Extracellular Domain of the Platelet-activating Receptor CLEC-2

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-A resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding.

Mutation of Tn5 Transposase β-Loop Residues Affects All Steps of Tn5 Transposition: The Role of Conformational Changes in Tn5 Transposition

X-ray cocrystal structures of Tn5 transposase (Tnp) bound to its 19 base pair (bp) recognition end sequence (ES) reveal contacts between a beta-loop (amino acids 240-260) and positions 3, 4, 5, and 6 of the ES. Here, we show that mutations of residues in this loop affect both in vivo and in vitro transposition. Most mutations are detrimental, whereas some mutations at position 242 cause hyperactivity. More specifically, mutations to the beta-loop affect every individual step of transposition tested. Mutants performing in vivo and in vitro transposition less efficiently also form fewer synaptic complexes, whereas hyperactive Tnps form more synaptic complexes. Surprisingly, two hypoactive mutations, K244R and R253L, also affect the cleavage steps of transposition with a much more dramatic effect on the second double end break (DEB) complex formation step, indicating that the beta-loop likely plays an important roll in positioning the substrate DNA within the catalytic site. Finally, all mutants tested decrease efficiency of the final transposition step, strand transfer. A disparity in cleavage rate constants in vitro for mutants with changes to the proline at position 242 on transposons flanked by ESs differing in the orientation of the A-T base pair at position 4 allows us to postulate that P242 contacts the position 4 nucleotide pair. On the basis of these data, we propose a sequential model for end cleavage in Tn5 transposition in which the uncleaved PEC is not symmetrical, and conformational changes are necessary between the first and second cleavage events and also for the final strand transfer step of transposition.

Mutational and bioinformatics analysis of proline‐ and glycine‐rich motifs in vesicular acetylcholine transporter

The vesicular acetylcholine transporter (VAChT) contains six conserved sequence motifs that are rich in proline and glycine. Because these residues can have special roles in the conformation of polypeptide backbone, the motifs might have special roles in conformational changes during transport. Using published bioinformatics insights, the amino acid sequences of the 12 putative, helical, transmembrane segments of wild-type and mutant VAChTs were analyzed for propensity to form non-alpha-helical conformations and molecular notches. Many instances were found. In particular, high propensity for kinks and notches are robustly predicted for motifs D2, C and C'. Mutations in these motifs either increase or decrease Vmax for transport, but they rarely affect the equilibrium dissociation constants for ACh and the allosteric inhibitor, vesamicol. The near absence of equilibrium effects implies that the mutations do not alter the backbone conformation. In contrast, the Vmax effects demonstrate that the mutations alter the difficulty of a major conformational change in transport. Interestingly, mutation of an alanine to a glycine residue in motif C significantly increases the rates for reorientation across the membrane. These latter rates are deduced from the kinetics model of the transport cycle. This mutation is also predicted to produce a more flexible kink and tighter tandem notches than are present in wild-type. For the full set of mutations, faster reorientation rates correlate with greater predicted propensity for kinks and notches. The results of the study argue that conserved motifs mediate conformational changes in the VAChT backbone during transport.

Lipid–protein modifications during ascorbate‐Fe2+ peroxidation of photoreceptor membranes: protective effect of melatonin

The rod outer segment (ROSg) membranes are essentially lipoprotein complexes. Rhodopsin, the major integral protein of ROSg, is surrounded by phospholipids highly enriched in docosahexaenoic acid (22:6 n3). This fluid environment plays an important role for conformational changes after photo-activation. Thus, ROSg membranes are highly susceptible to oxidative damage. Melatonin synthesized in the pineal gland, retina and other tissues is a free radical scavenger. The principal aim of this work was to study the changes in the ROSg membranes isolated from bovine retina submitted to nonenzymatic lipid peroxidation (ascorbate-Fe2+ induced), during different time intervals (0-180 min). Oxidative stress was monitored by increase in the chemiluminescence and fatty acid alterations. In addition we studied the in vitro protective effect of 5 mm melatonin. The total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of melatonin. The docosahexaenoic acid content decreased considerably when the membranes were exposed to oxidative damage. This reduction was from 35.5 +/- 2.9% in the native membranes to 12.65 +/- 1.86% in those peroxidized during 180 min. In the presence of 5 mm melatonin we observed a content preservation of 22:6 n3 (23.85 +/- 2.77%) at the same time of peroxidation. Simultaneously the alterations of membrane proteins under oxidative stress were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Loss of protein sulfhydryl groups and increased incorporation of carbonyl groups were utilized as biomarkers of protein oxidation. In membranes exposed to Fe2+ -ascorbate, we observed a decrease of protein thiols from 50.9 +/- 3.38 in native membranes to 1.72 +/- 2.81 nmol/mg of protein after 180 min of lipid peroxidation associated with increased incorporation of carbonyl groups into proteins from 7.20 +/- 2.50 to 12.50 +/- 1.12 nmol/mg of protein. In the SDS-PAGE we observed a decrease in the content of all the proteins, mainly rhodopsin, as a consequence of peroxidation. Melatonin, prevent both lipid peroxidation and protein oxidation.

Single-molecule fluorescence studies of protein folding and conformational dynamics.

1.1. Aims of This Review Single-molecule measurements provide unique information on heterogeneous populations of molecules: They give access to the complete distribution of observables (rather than only their first moments), allow discrimination between static and dynamic heterogeneity of their properties, and enable the detection of rare events or a succession of events hidden by ensemble averaging and the impossibility to synchronize molecules.1–8 Single-molecule methods have now pervaded several disciplines, in particular chemistry, evolving from a stage of proof-of-principle experiments to decisive research and discovery tools. A literature database search with the keyword “single molecule” gave over 5000 references at the time of this writing. This renders the prospect of an exhaustive discussion of current single-molecule work rather daunting. It proves, however, that single-molecule methods have gained the status of established techniques in various scientific fields and continue to propagate to new ones. Therefore, it seemed appropriate for a review of single-molecule methods in chemistry to provide a rapid description of the main technical approaches and focus on a few illustrative examples of their elucidative power. Even such an endeavor would have resulted in a heteroclite description of research on topics as diverse as quantum electrodynamics, low temperature and room temperature experiments on nanoparticles and organic or biological molecules, micromechanical manipulation, or fluorescence spectroscopy, among many others. Such an accumulation would have been of little interest, once the basic principles underlying each technique had been explained. It seemed, therefore, more appropriate to describe applications of a unique set of methods (fluorescence spectroscopy) to biochemical questions and, more specifically, the elucidation of protein structure, dynamics, and function. Protein structure and function are intimately related, and a large amount of single-molecule studies have been performed to elucidate the nature and role of conformational changes in protein or enzyme functions. These questions are best studied when methods have been validated on model systems, and we will delve into some simple examples of such systems to illustrate concepts, which are used in more sophisticated and ambitious studies. Another important aspect of protein science is the mechanism of protein structure formation (and loss thereof), i.e., protein folding and unfolding. Single-molecule methods have begun to yield very interesting results in this domain, and undoubtedly, more will follow. We have thus made this promising field one of the focuses of our review. The review is organized as follows. We will first define the questions that have been studied so far at the ensemble level and that are now being addressed with single-molecule fluorescence spectroscopy. The next section presents a summary of basic concepts of fluorescence spectroscopy and briefly reviews recent developments in single-molecule analysis, to serve as a glossary for all experimental approaches described throughout the remainder of this review. We then turn to applications of single-molecule fluorescence resonance energy transfer (FRET) to study polypeptide chain collapse in small single-domain proteins under equilibrium conditions. We provide some examples on how to extract dynamic information from single molecules, namely, distance distributions within conformational subpopulations of proteins in the framework of protein folding and in enzymes. These aspects are divided into two parts: studies based on FRET and studies relying on fluorescence quenching. The last part of this review addresses recent studies of protein folding dynamics under nonequilibrium conditions. We conclude with general remarks and an overview of future prospects of these methods.

Structural analysis of Thr342 mutants of soybean β-amylase: the role of conformational changes of two loops in the catalytic mechanism

Structural analysis of Thr342 mutants of soybean β-amylase: the role of conformational changes of two loops in the catalytic mechanism

Synthesis of an N-acyl sulfamate analog of luciferyl-AMP: A stable and potent inhibitor of firefly luciferase

In the first of two half-reactions resulting in the emission of visible light, firefly luciferase forms luciferyl-adenylate from its natural substrates beetle luciferin and Mg-ATP. The acyl-adenylate is subsequently oxidized producing the light emitter oxyluciferin in an electronically excited state. In vitro, under mild conditions of temperature and pH, the acyl-adenylate intermediate is readily hydrolyzed and susceptible to oxidation. We report here the multi-step synthesis and physical and enzymatic characterization of an N-acyl sulfamate analog of luciferyl-adenylate, 5′- O-[( N-dehydroluciferyl)-sulfamoyl]-adenosine (compound 5). This represents the first example of a stable and potent ( K i = 340 nM) reversible inhibitor of firefly luciferase activity based on the structure of the natural acyl-adenylate intermediate. Additionally, we present the results of limited proteolysis studies that demonstrate that the binding of the novel acyl-adenylate analog protects luciferase from proteolysis. The findings presented here are interpreted in the context of the hypothesis that luciferase and the other enzymes in a large superfamily of adenylate-forming proteins adopt two conformations to catalyze two different partial reactions. We anticipate that the novel N-acyl sulfamate analog will be a valuable reagent in future studies designed to elucidate the role of conformational changes in firefly luciferase catalyzed bioluminescence.

De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly–Asp–Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.

Excited state relaxation of 9-(4-diethylaminostyryl)-acridine

Excited state dynamics of neutral and protonated 9-(4-diethylaminostyryl)-acridine was investigated by means of picosecond fluorescence and pump-and-probe absorption techniques. Both forms show complex transient absorption spectra evolution on a ps time scale and multicomponental transient absorption relaxation, strongly dependent on the probe wavelength. The protonated form shows very weak fluorescence and its relaxation kinetics slows down in more viscous solvents indicating that conformational changes of molecules take place during relaxation of the excited state and form a significant energy deactivation channel. During the relaxation of the neutral molecules two fluorescent intermediates with lifetimes of 10 and 90 ps are formed. Relaxation dynamics of the neutral molecules is independent of the solvent viscosity excluding role of conformational changes.

Conformational change in substrate binding, catalysis and product release: an open and shut case?

The role of conformational change in substrate binding, catalysis and product release is reviewed for 11 enzymes, for which crystal structures are available for the apo, substrate- and product-bound states. The extent of global conformational changes is measured, and the movements of the functional regions involved in catalysis and ligand binding are compared to the rest of the structure. We find that most of these enzymes undergo relatively small amounts of conformational change and particularly small changes in catalytic residue geometry, usually less than 1 Å. In some enzymes there is significant movement of the binding residues, usually on surface loops.