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At least 19 recordsLinked to original sources

PubMed2004

Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase: computational studies of the ground state, transition state, and intermediate.

Peptide amidase (Pam), a hydrolytic enzyme that belongs to the amidase signature (AS) family, selectively catalyzes the hydrolysis of the C-terminal amide bond (CO-NH(2)) of peptides. The recent availability of the X-ray structures of Pam, fatty acid amide hydrolase, and malonamidase E2 has led to the proposal of a novel Ser-Ser-Lys catalytic triad mechanism for the amide hydrolysis by the AS enzymes. The molecular dynamics (MD) simulations using the CHARMM force field were performed to explore the catalytic mechanism of Pam. The 1.8 A X-ray crystal structure of Pam in complex with the amide analogue of chymostatin was chosen for the initial coordinates for the MD simulations. The five systems that were investigated are as follows: (i) enzyme.substrate with Lys123-NH(2), (ii) enzyme.substrate with Lys123-NH(3)(+), (iii) enzyme.substrate with Lys123-NH(3)(+) and Ser226-O(-), (iv) enzyme.transition state, and (v) enzyme.tetrahedral intermediate. Our data support the presence of the hydrogen bonding network among the catalytic triad residues, Ser226, Ser202, and Lys123, where Ser226 acts as the nucleophile and Ser202 bridges Ser226 and Lys123. The MD simulation supports the catalytic role of the crystallographic waters, Wat1 and Wat2. In all the systems that have been studied, the backbone amide nitrogens of Asp224 and Thr223 create an oxyanion hole by hydrogen bonding to the terminal amide oxygen of the substrate, and stabilize the oxyanion tetrahedral intermediate. The results from both our computational investigation and previously published experimental pH profile support two mechanisms. In a mechanism that is relevant at lower pH, the Lys123-NH(3)(+)-Ser202 dyad provides structural support to the catalytic residue Ser226, which in turn carries out a nucleophilic attack at the substrate amide carbonyl in concert with Wat1-mediated deprotonation and stabilization of the tetrahedral transition state by the oxyanion hole. In the mechanism operating at higher pH, the Lys123-NH(2)-Ser202 catalytic dyad acts as a general base to assist addition of Ser226 to the substrate amide carbonyl. The results from the MD simulation of the tetrahedral intermediate state show that both Ser202 and Lys123 are possible candidates for protonation of the leaving group, NH(2), to form the acyl-enzyme intermediate.

Amidohydrolases↗

PubMed2003

Conformations of benzene- and dibenzo[a,l]pyrene diol epoxides studied by density functional theory: ground states, transition states, dynamics, and solvent effects.

The (-)-anti- and (+)-syn-diol epoxides of dibenzo[a,l]pyrene (DBPDE, 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene) and the stereochemically corresponding benzene diol epoxides (BDE, 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrobenzene) have been studied by density functional theory (DFT) to determine the structures, energies, dynamics, thermal properties, and solvent effects on the different conformers. The smaller BDE is used as a model compound for studies of transitions between diequatorial and diaxial conformations of the hydroxyl groups. It was found that DBPDE is distorted due to overcrowding in the fjord region and that the arene oxide prefers to be on the same side of the saturated ring as the distal ring ("in") in most stereoisomeric states. For the anti-diastereomer, a diequatorial orientation of the hydroxyl groups is preferred, while the orientation preference in the syn-diastereomer seems to depend on the solvent and the in/out conformation. Transition states for the interconversions between in and out conformations of DBPDE as well as between diequatorial and diaxial conformations on BDE have been found, and transition rates have been estimated by transition state theory. The barriers are found to be moderate, the highest being 9.6 kcal/mol. Solvent effects as well as zero-point vibrational energy and thermal effects were included and found to be significant in some cases. The results presented here are in agreement with previous experimental studies.

Benzopyrans↗

PubMed2005

Enzymatic transition states and transition state analogues.

Transition states are the balance point of catalysis. Bonds are partially made and/or broken at the transition state, and the energy of the extended system provides near-equal probability that the system forms products or reverts to reactants. Enzymatic catalytic sites provide dynamic electronic environments that increase the probability that the transition state will be formed. Alignment of reactants in the Michaelis complex and motion of the catalytic site architecture are necessary to achieve the transition state. Transition state lifetimes are a fraction of a picosecond, preventing chemical equilibrium in extended covalent systems. Thus, dynamic descriptions of enzymatic transition states are required. Stable analogues similar to the transition state capture dynamic excursions that generate the transition state and convert them into thermodynamic binding energy. These analogues bind with extraordinary affinity relative to reactants.

Animals↗

PubMed1998

Enzymatic transition states and transition state analog design.

All chemical transformations pass through an unstable structure called the transition state, which is poised between the chemical structures of the substrates and products. The transition states for chemical reactions are proposed to have lifetimes near 10(-13) sec, the time for a single bond vibration. No physical or spectroscopic method is available to directly observe the structure of the transition state for enzymatic reactions. Yet transition state structure is central to understanding catalysis, because enzymes function by lowering activation energy. An accepted view of enzymatic catalysis is tight binding to the unstable transition state structure. Transition state mimics bind tightly to enzymes by capturing a fraction of the binding energy for the transition state species. The identification of numerous transition state inhibitors supports the transition state stabilization hypothesis for enzymatic catalysis. Advances in methods for measuring and interpreting kinetic isotope effects and advances in computational chemistry have provided an experimental route to understand transition state structure. Systematic analysis of intrinsic kinetic isotope effects provides geometric and electronic structure for enzyme-bound transition states. This information has been used to compare transition states for chemical and enzymatic reactions; determine whether enzymatic activators alter transition state structure; design transition state inhibitors; and provide the basis for predicting the affinity of enzymatic inhibitors. Enzymatic transition states provide an understanding of catalysis and permit the design of transition state inhibitors. This article reviews transition state theory for enzymatic reactions. Selected examples of enzymatic transition states are compared to the respective transition state inhibitors.

Catalysis↗

PubMed2005

Analysis of the pH-dependent folding and stability of histidine point mutants allows characterization of the denatured state and transition state for protein folding.

pH-Dependent studies of the folding kinetics and stability of a set of His to Gln point mutants were used to characterize the denatured state and transition state ensembles for the C-terminal domain of the ribosomal protein L9 (CTL9). CTL9 contains three histidine residues, two of which, H106 and H134, are buried in the native state, while the third, H144, is more exposed. Comparison of the pH-dependent stability calculated using the Tanford-Wyman linkage relationship to the measured values demonstrates that the apparent pK(a) values of the three histidine residues are not significantly perturbed in the denatured state ensemble. Kinetic measurements show that mutation of H134 has a larger effect on the folding process than does mutation of H106 and H144. The Phi-value for H134 is significantly larger than the Phi-values for the other histidine residues, which are near zero at both pH 5.45 and pH 8.0. The Phi-value for H134 is higher, 0.55, at pH 8.0 than at pH 5.45, 0.39. At pH 5.45, H134 is protonated in the unfolded state but deprotonated in the native state, while at pH 8.0 it is deprotonated in both. There is an excellent linear relationship between stability (logK) and folding rates (logk(f)) over the range of pH 5-9 for all mutants. From these plots, the ratio of DeltaQ( not equal)/DeltaQ can be calculated for each mutant. DeltaQ( not equal) is the difference in the number of protons bound to the transition state and to the unfolded state, while DeltaQ represents the difference between folded and denatured state. The linear plots indicate that the relative position of the transition state ensemble as judged by DeltaQ( not equal)/DeltaQ is independent of pH. The linkage analysis is consistent with the Phi-value analysis, showing that H134 is the most critical contributor to the development of pH-dependent interactions, including desolvation effects in the transition state ensemble.

Circular Dichroism↗

PubMed1985

Protein structural changes accompanying formation of enzymatic transition states: tryptophan environment in ground-state and transition-state analogue complexes of adenosine deaminase.

The accessibility of protein tryptophan fluorescence to the quenching agent acrylamide has been studied in adenosine deaminase and in binary complexes of the enzyme with ground-state or transition-state analogues. Although the enzyme contains three tryptophan residues, Stern-Volmer plots are linear with all the fluorescence quenchable at high acrylamide concentrations. Tryptophan fluorescence is less easily quenched in the binary complexes than in the free enzyme, indicating a decrease in the accessibility of these residues. The greatest decrease in accessibility is found for the transition-state analogue complexes. Although the affinities of the transition-state analogues studied span a range of 10(6), the Stern-Volmer constants of the complexes are the same within experimental error. Thus, as measured by this technique, changes in enzyme conformation accompanying formation of these complexes are similar for all transition-state analogues. Resonance energy transfer from tryptophan as donor to ligand as acceptor successfully explains the differing abilities of ligands to quench the enzyme's intrinsic fluorescence upon formation of complexes in the absence of acrylamide. On the basis of Forster distance calculations, it is likely that the residues partially quenched upon formation of transition-state analogue complexes are distant from the active site.

Adenosine Deaminase↗

PubMed2003

Enzymatic transition state poise and transition state analogues.

The development of kinetic isotope effect methods for enzymatic reactions has resulted in the systematic determination of enzymatic transition state structure for several distinct chemical reaction mechanisms. Although it is early in the experimental development of the method, examples of concerted nucleophilic displacements (A(N)D(N) or S(N)2), aromatic nucleophilic displacements (A(N)D(N) or S(N)Ar), and both concerted and stepwise dissociative nucleophilic displacements (D(N)A(N) and D(N)A(N); S(N)1 reactions) have been exemplified. The transition state for each reaction exhibits a characteristic extent of bond-breaking and bond-making, defined here as transition state poise. Thus, concerted nucleophilic displacements (S(N)2 or D(N)A(N)) exhibit various extents of residual bond order to the leaving group and to the attacking nucleophile at the transition state. Aromatic nucleophilic displacements reach their rate-limiting transition states before or after formation of the tetrahedral intermediate. Several concerted, symmetric nucleophilic displacements at carbon have been described. Enzymatic transition state poise is summarized in a single diagram of bond orders using the terminology of Jencks. The analysis reveals enzymatic contributions to transition state poise, provides precedent for assignment of reaction types, and summarizes the current status of the experimental characterization of enzymatic transition states. Binding strengths of transition state analogues are readily correlated with transition state poise.

Animals↗

PubMed2004

Transition state dynamics of N2O4 <==> 2NO2 in liquid state.

Transition state dynamics of dissociation and association reactions N2O4 <==> 2NO2 in liquid state are studied by classical molecular dynamics simulations of reactive liquid NO(2) at 298 K. An OSPP+LJ potential between NO(2) molecules proposed in paper I [J. Chem. Phys. 115, 10852 (2001)], which takes into account the orientational sensitivity of the chemical bond, has been used in the simulation. The trajectory and energy evolution of various reactions are studied in the OSPP+LJ liquid, which reproduces both the observed liquid phase equilibrium constant and Raman band shape of the dissociation mode. It is found that a NO(2) pair in reactive liquid NO(2) is bound when E(T)<0 and dissociates when E(T)>0, and the dissociation of a reactant pair occurs when the transition state (TS) surface of E(T)=0 is crossed from negative to positive, where E(T) is the sum of the potential and kinetic energies of intermolecular motion of the pair. Two types of dissociation are found depending on the source of energy for dissociation; the first type D is the dissociation via collisional activation of the reactive mode by solvent molecules, and the second type T is the dissociation via bond transfer from a dimer to a monomer NO(2) through the TS of NO(2) trimer. It is concluded that the type T dissociation is found to be much more probable than the type D dissociation because of easy energy conservation. The reactant experiences the TS of NO(2) trimer for a long time (1-10 ps) in NO(2) mediated bond transfer reactions, and crossing and recrossing trajectories and dynamics in the TS neighborhood are studied.

Journal Article↗

PubMed2005

Ground-state, transition-state, and metal-cation effects of the 2-hydroxyl group on beta-D-galactopyranosyl transfer catalyzed by beta-galactosidase (Escherichia coli, lac Z).

Substitution of the C2-OH group by C2-H at 4-nitrophenyl-beta-d-galactopyranoside to give 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside causes (1) a change in the rate-determining step for beta-galactosidase-catalyzed sugar hydrolysis from formation to breakdown of a covalent intermediate; (2) a 14 000-fold decrease in the second-order rate constant k(3)/K(d) for enzyme-catalyzed transfer of the beta-d-galactopyranosyl group from the substrate to form a covalent adduct to the enzyme; and (3) a larger 320 000-fold decrease in the first-order rate constant k(s) for hydrolysis of this covalent adduct. Only a small fraction (ca. 7%) of the 2-OH substituent effect is expressed in the ground-state Michaelis complex, so that the (apparent) strong interactions between the enzyme and 2-OH group that stabilize the transition state for beta-d-galactopyranosyl transfer only develop upon moving from the Michaelis complex to the transition state. Mg(2+) activates beta-galactosidase for cleavage of both 4-nitrophenyl-beta-d-galactopyranoside and 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside. This suggests that Mg(2+) activation does not involve interactions with the 2-OH group. The removal of Mg(2+) from beta-galactosidase causes a change in the rate-determining step for enzyme-catalyzed hydrolysis of 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside from breakdown to formation of the covalent intermediate. The observed 2-OH effect would require a very large (10-11 kcal/mol) stabilization of the transition state for beta-d-galactopyranosyl group transfer to water by interactions between beta-galactosidase and the neutral 2-OH group. We suggest that the apparent effect of the neutral substituent is more simply rationalized by ionization of the 2-OH to form a 2-O(-) anion, which provides effective electrostatic stabilization of the cationic transition state for glycoside cleavage at an active site of relatively low dielectric constant.

Deoxy Sugars↗

PubMed2001

Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5'-phosphate decarboxylase: a role for substrate destabilization?

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of strong interactions between enzyme residues and functional groups of this ligand. Enzyme contacts with the phosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least 16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reaction catalyzed by yeast ODCase. In addition to these enzyme-ligand contacts, active site residues contributed by both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety. These involve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectively. To evaluate the contributions of these ribofuranosyl contacts to ground state and transition state stabilization, Thr-100 and Asp-37 were each mutated to alanine. Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K(m) value of the T100A enzyme by 60-fold and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude. Upon removal of the complementary hydroxymethyl group from the enzyme, little further reduction in k(cat)/K(m) for 2'-deoxyOMP was observed. To assess the contribution made by contacts involving both ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured. The value of k(cat)/K(m) for this enzyme-substrate pair was 170 M(-1) s(-1), representing a decrease of more than 7.6 kcal/mol of binding free energy in the transition state. To the extent that electrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complex plays a major role in catalysis.

Alanine↗

PubMed2003

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease.

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Brønsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.

Amino Acid Sequence↗

PubMed1993

Correlation of the molecular electrostatic potential surface of an enzymatic transition state with novel transition-state inhibitors.

The transition state stabilized by nucleoside hydrolase from Crithidia fasciculata is characterized by nearly complete glycosidic bond cleavage and oxycarbonium character in the ribosyl group [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. The electrostatic potential surface of the transition state provides detailed information which should be useful in the design of transition-state analogues [Horestein, B. A., & Schramm, V. L. (1993) Biochemistry 32, 7089-7097]. The electrostatic potential surface of inosine at the transition state contains a distributed positive charge resulting from the oxycarbonium ion character of the ribosyl ring. The ribosyl ring pucker is 3'-exo as a result of the near sp2 hybridization at Cl' of the ribose ring. A series of transition-state analogues have been synthesized which incorporate single or combined features of the transition state. Each feature of the transition state was analyzed for its contribution to binding energy. Kinetic inhibition constants correlate with the similarity of the inhibitor to the experimentally determined transition-state structure. Dissociation constants for the substrate and products of the reaction of inosine, hypoxanthine, and ribose are 380, 6200, and 700 microM, respectively. A transition-state analogue was synthesized which contains the required hydroxyl groups of the ribose ring, the positive charge feature of the oxycarbonium ion, and a hydrophobic mimic of the purine ring. The inhibitor 1(S)-phenyl-1,4-dideoxy-1,4-iminoribitol acts as a competitive inhibitor with respect to inosine with a dissociation constant of 0.17 microM. In addition, the inhibitor exhibits slow-onset inhibition which provides a final equilibrium dissociation constant of approximately 0.03 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Pre-steady-state transition-state analysis of the hydrolytic reaction catalyzed by purine nucleoside phosphorylase.

The slow hydrolytic reaction catalyzed by calf spleen purine nucleoside phosphorylase [Kline, P. C., & Schramm, V. L. (1992) Biochemistry 31, 5964-5973] has been investigated using pre-steady-state kinetic isotope effects and solvolysis studies. The stoichiometric reaction between enzyme and inosine forms 1 mol of free ribose per trimer of purine nucleoside phosphorylase and a tightly bound complex of enzyme and hypoxanthine. The experimental kinetic isotope effects from [1'-3H]-, [2'-3H]-, [4'-3H]-, [5'-3H]-, [1'-14C]-, and [9-15N]inosine are 1.151 +/- 0.004, 1.145 +/- 0.003, 1.006 +/- 0.004, 1.028 +/- 0.005, 1.045 +/- 0.005, and 1.000 +/- 0.005, respectively, for the pre-steady-state conditions. Substrate trapping experiments demonstrated that there is no detectable forward commitment to catalysis for inosine hydrolysis. In contrast, bound inosine is 2.1 times more likely to form product than to dissociate when the enzyme-inosine complex is exposed to saturating PO4. The lack of an observed 9-15N isotope effect is consistent with an internal equilibrium between enzyme-inosine and the enzyme-hypoxanthine-ribose complex in which N9 of hypoxanthine is protonated. The equilibrium occurs as a consequence of slow product release and tightly bound hypoxanthine (Kd = 1.3 x 10(-12) M). This internal equilibrium has a minimal effect on the intrinsic kinetic isotope effects from ribose since equilibrium isotope effects for conversion of inosine to ribose are near unity. When the single-turnover hydrolytic reaction was accomplished in 20% methanol, approximately 85% of the product sugar was 1-methylribose. Under these conditions, the anion-binding pocket fills with solvent which competes for the oxocarbenium ion of inosine formed at the transition state. In the presence of arsenate, no methanolysis of inosine occurs [Kline, P. C., & Schramm, V. L. (1993) Biochemistry 32, 13212-13219]. The results define a transition state with oxocarbenium ion character and weak participation of the attacking solvent nucleophile. Electrostatic potential surfaces of the transition states indicate that arsenate anion is more effective in neutralizing the oxocarbenium ion than is H2O.

Adenosine↗

PubMed2006

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure.

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.

Hydrogen-Ion Concentration↗

PubMed2006

Do proteins with similar folds have similar transition state structures? A diffuse transition state of the 169 residue apoflavodoxin.

Apoflavodoxin from Anabaena PCC 7119 is a 169 residue globular protein of known structure and energetics. Here, we present a comprehensive Phi-value analysis to characterize the structure of its transition state. A total of 34 non-disruptive mutations are made throughout the structure and a range of Phi-values from zero to one are observed. In addition, a small set of eight aliphatic small-to-large mutations have been introduced in the hydrophobic core of the protein and they have been analyzed to investigate the feasibility of stabilizing the unfolding transition state by creating new non-native interactions. We find that the transition state of apoflavodoxin (so far the largest protein subjected to Phi-analysis) is diffuse and that it can be stabilized by unspecific hydrophobic interactions that can speed up the folding reaction. The data gathered on the apoflavodoxin transition state are compared with results from experimental studies in other proteins to revisit the relationship between the native state topology and transition state structure.

Anabaena↗

PubMed2001

Energetics of coiled coil folding: the nature of the transition states.

Coiled coils are simple models for studying the association of two polypeptide chains to form a folded protein. Previous work has shown that the folding of a coiled coil can be described by a two-state transition between two unfolded monomeric peptide chains and a folded coiled coil dimer. Here we report the thermodynamic activation parameters for the folding and unfolding of two unrelated coiled coils: C62GCN4 and A(2). C62GCN4 corresponds to the 62 C-terminal residues of yeast transcription factor GCN4. The peptide forms a dimeric coiled coil through its 33 C-terminal residues. A(2) is a designed 30-residue dimeric coiled coil whose folding is induced by low pH [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Folding and unfolding were assessed under identical native buffer conditions so that the microscopic reversibility applied and the transition state was the same for folding and unfolding. The time course of folding was followed from the self-quenching of a C-terminal fluorescent label (Texas Red). The overall folding of both peptides is enthalpy-driven and opposed by a loss of entropy. The main energetic changes occur after the system has passed the transition state. In the folding of C62GCN4, only 10-20% of the heat capacity change is attained between the monomeric state and the dimeric transition state. For coiled coil A(2), the fractional heat capacity change preceding the transition state is 30-40%. The results indicate that the activated states of folding of coiled coils are not well structured and differ considerably from the folded coiled coil conformation. These findings are in agreement with a rate-limiting transition state in which the coiled coil helices and the hydrophobic coiled coil interface are poorly developed.

Amino Acid Motifs↗

PubMed2003

Structural analysis of the rate-limiting transition states in the folding of Im7 and Im9: similarities and differences in the folding of homologous proteins.

The bacterial immunity proteins Im7 and Im9 fold with mechanisms of different kinetic complexity. Whilst Im9 folds in a two-state transition at pH 7.0 and 10 degrees C, Im7 populates an on-pathway intermediate under these conditions. In order to assess the role of sequence versus topology in the folding of these proteins, and to analyse the effect of populating an intermediate on the landscape for folding, we have determined the conformational properties of the rate-limiting transition state for Im9 folding/unfolding using Phi(F)-value analysis and have compared the results with similar data obtained previously for Im7. The data show that the rate-limiting transition states for Im9 and Im7 folding/unfolding are similar: both are compact (beta(T)=0.94 and 0.89, respectively) and contain three of the four native helices docked around a specific hydrophobic core. Significant differences are observed, however, in the magnitude of the Phi(F)-values obtained for the two proteins. Of the 20 residues studied in both proteins, ten have Phi(F)-values in Im7 that exceed those in Im9 by more than 0.2, and of these five differ by more than 0.4. The data suggest that the population of an intermediate in Im7 results in folding via a transition state ensemble that is conformationally restricted relative to that of Im9. The data are consistent with the view that topology is an important determinant of folding. Importantly, however, they also demonstrate that while the folding transition state may be conserved in homologous proteins that fold with two and three-state kinetics, the population of an intermediate can have a significant effect on the breadth of the transition state ensemble.

Alanine↗

PubMed2006

Butatrienes as extended alkenes: barriers to internal rotation and substitution effects on the stabilities of the ground states and transition states.

The barriers to internal rotation of methylated, ethynylated, and vinylated butatrienes and alkenes were calculated at the CASPT2/6-31G(d)//B3LYP/6-31G(d) level. Calculated butatriene rotational barriers are lower than those of analogous alkenes, but there is a larger variance in rotational barrier for alkenes than for butatrienes. The barriers to rotation were analyzed by isodesmic equations designed to estimate the substituent effects in the ground (GS) and transition (TS) states individually. The GSs of both series are stabilized to roughly the same extent. In contrast, the TSs of butatrienes are more stabilized overall than those of alkenes. Much of the stabilization in the TS of butatrienes comes from the internal triple bond and not from the substituent. Estimation of the substituent stabilization alone reveals the TSs of ethylenes to be more stabilized by substitution than butatrienes.

Journal Article↗