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PubMed2005

Role of transmembrane segment 10 in efflux mediated by the staphylococcal multidrug transport protein QacA.

The staphylococcal multidrug exporter QacA confers resistance to a wide range of structurally dissimilar monovalent and bivalent cationic antimicrobial compounds. To understand the functional importance of transmembrane segment 10, which is thought to be involved in substrate binding, cysteine-scanning mutagenesis was performed in which 35 amino acid residues in the putative transmembrane helix and its flanking regions were replaced in turn with cysteine. Solvent accessibility analysis of the introduced cysteine residues using fluorescein maleimide indicated that transmembrane segment 10 of QacA contains a 20-amino-acid hydrophobic core and may extend from Pro-309 to Ala-334. Phenotypic analysis and fluorimetric transport assays of these mutants showed that Gly-313 is important for the efflux of both monovalent and bivalent cationic substrates, whereas Asp-323 is only important for the efflux of bivalent substrates and probably forms part of the bivalent substrate-binding site(s) together with Met-319. Furthermore, the effects of N-ethyl-maleimide treatment on ethidium and 4',6-diamidino-2-phenylindole export mediated by the QacA mutants suggest that the face of transmembrane segment 10 that contains Asp-323 may also be close to the monovalent substrate-binding site(s), making this helix an integral component of the QacA multidrug-binding pocket.

Amino Acid Sequence↗

PubMed2005

The solvation interface is a determining factor in peptide conformational preferences.

The 21 residue polyalanine-based F(s) peptide was studied using thousands of long, explicit solvent, atomistic molecular dynamics simulations that reached equilibrium at the ensemble level. Peptide conformational preference as a function of hydrophobicity was examined using a spectrum of explicit solvent models, and the peptide length-dependence of the hydrophilic and hydrophobic components of solvent-accessible surface area for several ideal conformational types was considered. Our results demonstrate how the character of the solvation interface induces several conformational preferences, including a decrease in mean helical content with increased hydrophilicity, which occurs predominantly through reduced nucleation tendency and, to a lesser extent, destabilization of helical propagation. Interestingly, an opposing effect occurs through increased propensity for 3(10)-helix conformations, as well as increased polyproline structure. Our observations provide a framework for understanding previous reports of conformational preferences in polyalanine-based peptides including (i) terminal 3(10)-helix prominence, (ii) low pi-helix propensity, (iii) increased polyproline conformations in short and unfolded peptides, and (iv) membrane helix stability in the presence and absence of water. These observations provide physical insight into the role of water in peptide conformational equilibria at the atomic level, and expand our view of the complexity of even the most "simple" of biopolymers. Whereas previous studies have focused predominantly on hydrophobic effects with respect to tertiary structure, this work highlights the need for consideration of such effects at the secondary structural level.

Hydrophobic and Hydrophilic Interactions↗

PubMed1996

Molecular dynamics simulations of N-terminal peptides from a nucleotide binding protein.

Molecular dynamics (MD) simulations of N-terminal peptides from lactate dehydrogenase (LDH) with increasing length and individual secondary structure elements were used to study their stability in relation to folding. Ten simulations of 1-2 ns of different peptides in water starting from the coordinates of the crystal structure were performed. The stability of the peptides was compared qualitatively by analyzing the root mean square deviation (RMSD) from the crystal structure, radius of gyration, secondary and tertiary structure, and solvent accessible surface area. In agreement with earlier MD studies, relatively short (< 15 amino acids) peptides containing individual secondary structure elements were generally found to be unstable; the hydrophobic alpha 1-helix of the nucleotide binding fold displayed a significantly higher stability, however. Our simulations further showed that the first beta alpha beta supersecondary unit of the characteristic dinucleotide binding fold (Rossmann fold) of LDH is somewhat more stable than other units of similar length and that the alpha 2-helix, which unfolds by itself, is stabilized by binding to this unit. This finding suggests that the first beta alpha beta unit could function as an N-terminal folding nucleus, upon which the remainder of the polypeptide chain can be assembled. Indeed, simulations with longer units (beta-alpha-beta-alpha and beta-alpha-beta-alpha beta-beta) showed that all structural elements of these units are rather stable. The outcome of our studies is in line with suggestions that folding of the N-terminal portion of LDH in vivo can be a cotranslational process that takes place during the ribosomal peptide synthesis.

Amino Acid Sequence↗

PubMed2002

The treatment of solvation by a generalized Born model and a self-consistent charge-density functional theory-based tight-binding method.

We present a model to calculate the free energies of solvation of small organic compounds as well as large biomolecules. This model is based on a generalized Born (GB) model and a self-consistent charge-density functional theory-based tight-binding (SCC-DFTB) method with the nonelectrostatic contributions to the free energy of solvation modeled in terms of solvent-accessible surface areas (SA). The parametrization of the SCC-DFTB/GBSA model has been based on 60 neutral and six ionic molecules composed of H, C, N, O, and S, and spanning a wide range of chemical groups. Effective atomic radii as parameters have been obtained through Monte Carlo Simulated Annealing optimization in the parameter space to minimize the differences between the calculated and experimental free energies of solvation. The standard error in the free energies of solvation calculated by the final model is 1.11 kcal mol(-1). We also calculated the free energies of solvation for these molecules using a conductor-like screening model (COSMO) in combination with different levels of theory (AM1, SCC-DFTB, and B3LYP/6-31G*) and compared the results with SCC-DFTB/GBSA. To assess the efficiency of our model for large biomolecules, we calculated the free energy of solvation for a HIV protease-inhibitor complex containing 3,204 atoms using the SCC-DFTB/GBSA and the SCC-DFTB/COSMO models, separately. The computed relative free energies of solvation are comparable, while the SCC-DFTB/GBSA model is three to four times more efficient, in terms of computational cost.

HIV Protease↗

PubMed1994

Use of amino acid environment-dependent substitution tables and conformational propensities in structure prediction from aligned sequences of homologous proteins. II. Secondary structures.

A three-step method is presented to predict secondary structures of proteins, by utilizing aligned sequences of homologous proteins. Mean propensities and amino acid substitution patterns at a given site in the aligned sequences are first evaluated for four conformational states (i.e. alpha-helix, beta-strand, buried coil and exposed coil). Capping rules are applied in order to define boundaries of the secondary-structure segments more precisely. In the second step beta-strand is predicted by searching regions predicted as coil for the two patterns characteristic of alternating and fully buried beta-strands. The complete sequences of the solvent-accessibility classes predicted by substitution tables and propensities are also searched using Fourier transform methods for alpha-helical periodicity. After applying capping rules, the alpha-helices and beta-strands predicted in the second step replace, where appropriate, the conformational states predicted in the first step. Finally, in the third step, if one of the four conformational states is assigned to the residues at an equivalent site of aligned sequences in more than a given fraction of the proteins, such a state is reassigned to all the residues at that site. The method is applied to 13 protein families, which contain four folding types, alpha, beta, alpha/beta and alpha + beta. The accuracy of the prediction ranges from 60 to 79% (mean percentage over the 13 families is 69%). For comparison the Garnier-Osguthorpe-Robson (GOR) method is also applied to them. Although the mean prediction accuracy for the GOR method, 58%, can be improved to 63% by applying the second and third steps in this method, there remain four families with less than 55% accuracy. The mean accuracy is relatively higher and poor predictions are reduced in this method.

Amino Acid Sequence↗

PubMed2005

Identification of 1,2,3,4,5,6-hexabromocyclohexane as a small molecule inhibitor of jak2 tyrosine kinase autophosphorylation [correction of autophophorylation].

The commercially available Jak2 inhibitor, alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490), has been used extensively to study Jak2 kinase function. While alpha-cyano-3,4-dihydroxy-N-benzylcinnamide is a potent Jak2 inhibitor, it can inhibit a number of other kinase signaling pathways as well. To circumvent this problem, we sought to identify novel small molecule inhibitors of Jak2 tyrosine kinase activity. For this, we constructed a homology model of the Jak2 kinase domain and identified solvent accessible pockets on the surface of the structure. Using the DOCK program, we tested 6451 compounds of known chemical structure in silico for their ability to interact with a pocket positioned adjacent to the activation loop. We attained the top seven scoring compounds from the National Cancer Institute and tested their ability to inhibit Jak2 autophosphorylation in vitro. Using Western blot analysis, we found that one of the compounds, 1,2,3,4,5,6-hexabromocyclohexane, was able to potently, and directly, inhibit Jak2 autophosphorylation. Characterization of this compound revealed that it inhibits Jak2 tyrosine autophosphorylation in both a time- and concentration-dependent manner. It greatly reduced growth hormone-mediated Jak2 autophosphorylation but did not block autophosphorylation of the epidermal growth factor receptor. Furthermore, doses as high as 100 muM were not toxic to cells as measured by their ability to exclude propidium iodide. As such, we believe that this compound could serve as a lead compound for a new generation of Jak2 inhibitors and, perhaps, be useful in elucidating the mechanisms of Jak2 kinase function.

Animals↗

PubMed2003

Structural reorganization of proteins revealed by radiolysis and mass spectrometry: G-actin solution structure is divalent cation dependent.

The solution structures of isolated monomeric actins in their Mg(2+)-ATP and Ca(2+)-ATP bound forms and in complexes with gelsolin segment-1 have been probed using hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry analysis of 28 peptides containing 58 distinct reactive probe sites within actin were used to monitor conformational variations linked to divalent cation and gelsolin segment-1 binding. The solvent accessibilities of the probe sites, as measured by footprinting in solution for the Ca(2+)-G-actin and Mg(2+)-G-actin complexes with gelsolin segment-1, were consistent with available crystallographic data. This included a specific protection at the contact interface between the partners, as revealed by reduced reactivity of peptide 337-359 in the complex. Aside from the specific protection indicated previously, the oxidation rates for the reactive residues of the isolated Ca(2+)-G-actin were similar to those of the actin gelsolin segment-1 complexes; however, the reactivity of numerous residues in the isolated Mg(2+)-G-actin form was significantly reduced. Specifically, Mg(2+)-G-actin has a set of protected sites relative to Ca(2+)-G-actin that suggest a structural reorganization in subdomains 4 and 2 and a C-terminus more closely packed onto subdomain 1. These conformational variations for isolated Mg(2+)-G-actin provide a structural basis for its greater tendency to polymerize into filaments as compared to Ca(2+)-G-actin.

Actins↗

PubMed2001

Optimization of solvation models for predicting the structure of surface loops in proteins.

A novel procedure for optimizing the atomic solvation parameters (ASPs) sigma(i) developed recently for cyclic peptides is extended to surface loops in proteins. The loop is free to move, whereas the protein template is held fixed in its X-ray structure. The energy is E(tot) = E(FF)(epsilon = nr) + summation operator sigma(i)A(i), where E(FF)(epsilon = nr) is the force-field energy of the loop-loop and loop-template interactions, epsilon = nr is a distance-dependent dielectric constant, and n is an additional parameter to be optimized. A(i) is the solvent-accessible surface area of atom i. The optimal sigma(i) and n are those for which the loop structure with the global minimum of E(tot)(n, sigma(i)) becomes the experimental X-ray structure. Thus, the ASPs depend on the force field and are optimized in the protein environment, unlike commonly used ASPs such as those of Wesson and Eisenberg (Protein Sci 1992;1:227-235). The latter are based on the free energy of transfer of small molecules from the gas phase to water and have been traditionally combined with various force fields without further calibration. We found that for loops the all-atom AMBER force field performed better than OPLS and CHARMM22. Two sets of ASPs [based on AMBER (n = 2)], optimized independently for loops 64-71 and 89-97 of ribonuclease A, were similar and thus enabled the definition of a best-fit set. All these ASPs were negative (hydrophilic), including those for carbon. Very good (i.e., small) root-mean-square-deviation values from the X-ray loop structure were obtained with the three sets of ASPs, suggesting that the best-fit set would be transferable to loops in other proteins as well. The structure of loop 13-24 is relatively stretched and was insensitive to the effect of the ASPs.

Hydrogen↗

PubMedDate not supplied

N-(iodoacetyl)-p-phenylenediamine-EDTA: a reagent for high-efficiency incorporation of an EDTA-metal complex at a rationally selected site within a protein.

We have developed a highly efficient procedure to incorporate an EDTA:metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step one, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step two, we derivatized the resulting protein with N-(iodoacetyl)-p-phenylenediamine-EDTA:metal, a novel haloacetyl derivative of EDTA:metal. We have used this procedure to incorporate each of three EDTA:metal complexes at amino acid 2 of the helix-turn-helix motif of the sequence-specific DNA binding protein Cro: a radioactive and nucleolytic EDTA:metal complex (EDTA:55Fe), a radioactive EDTA:metal complex (EDTA:63Ni), and a fluorescent and heavy-atom EDTA:metal complex (EDTA:Eu). Incorporation of EDTA:metal was highly efficient (> 80% for EDTA:55Fe and EDTA:63Ni; 60% for EDTA:Eu) and highly site-specific (> 99%). We have analyzed DNA affinity cleaving by the Cro derivative having EDTA:55Fe at amino acid 2 of the helix-turn-helix motif. The Cro derivative cleaves DNA at base pairs -4 to 6 of the DNA half site in the protein-DNA complex, indicating that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -4 to 6 of the DNA half site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗

PubMed1992

Empirical solvation models in the context of conformational energy searches: application to bovine pancreatic trypsin inhibitor.

Continuum solvation models that estimate free energies of solvation as a function of solvent accessible surface area are computationally simple enough to be useful for predicting protein conformation. The behavior of three such solvation models has been examined by applying them to the minimization of the conformational energy of bovine pancreatic trypsin inhibitor. The models differ only with regard to how the constants of proportionality between free energy and surface area were derived. Each model was derived by fitting to experimentally measured equilibrium solution properties. For two models, the solution property was free energy of hydration. For the third, the property was NMR coupling constants. The purpose of this study is to determine the effect of applying these solvation models to the nonequilibrium conformations of a protein arising in the course of global searches for conformational energy minima. Two approaches were used: (1) local energy minimization of an ensemble of conformations similar to the equilibrium conformation and (2) global search trajectories using Monte Carlo plus minimization starting from a single conformation similar to the equilibrium conformation. For the two models derived from free energy measurements, it was found that both the global searches and local minimizations yielded conformations more similar to the X-ray crystallographic structures than did searches or local minimizations carried out in the absence of a solvation component of the conformational energy. The model derived from NMR coupling constants behaved similarly to the other models in the context of a global search trajectory. For one of the models derived from measured free energies of hydration, it was found that minimization of an ensemble of near-equilibrium conformations yielded a new ensemble in which the conformation most similar to the X-ray determined structure PTI4 had the lowest total free energy. Despite the simplicity of the continuum solvation models, the final conformation generated in the trajectories for each of the models exhibited some of the characteristics that have been reported for conformations obtained from molecular dynamics simulations in the presence of a bath of explicit water molecules. They have smaller root mean square (rms) deviations from the experimentally determined conformation, fewer incorrect hydrogen bonds, and slightly larger radii of gyration than do conformations derived from search trajectories carried out in the absence of solvent.

Animals↗

PubMed1996

Solvation energies of amino acid side chains and backbone in a family of host-guest pentapeptides.

Octanol-to-water solvation free energies of acetyl amino amides (Ac-X-amides) [Fauchère, J.L., & Pliska, V. (1983) Eur. J. Med. Chem. --Chim. Ther. 18,369] form the basis for computational comparisons of protein stabilities by means of the atomic solvation parameter formalism of Eisenberg and McLachlan [(1986) Nature 319, 199]. In order to explore this approach for more complex systems, we have determined by octanol-to-water partitioning the solvation energies of (1) the guest (X) side chains in the host-guest pentapeptides AcWL-X-LL, (2) the carboxy terminus of the pentapeptides, and (3) the peptide bonds of the homologous series of peptides AcWLm (m = 1-6). Solvation parameters were derived from the solvation energies using estimates of the solvent-accessible surface areas (ASA) obtained from hard-sphere Monte Carlo simulations. The measurements lead to a side chain solvation-energy scale for the pentapeptides and suggest the need for modifying the Asp, Glu, and Cys values of the "Fauchère-Pliska" solvation-energy scale fro the Ac-X-amides. We find that the unfavorable solvation energy of nonpolar residues can be calculated accurately by a solvation parameter of 22.8 +/- 0.8 cal/mol/A2, which agrees satisfactorily with the AC-X-amide data and thereby validates the Monte Carlo ASA results. Unlike the Ac-X-amide data, the apparent solvation energies of the uncharged polar residues are also largely unfavorable. This unexpected finding probably results, primarily, from differences in conformation and hydrogen bonding in octanol and buffer but may also be due to the additional flaking peptide bonds of the pentapeptides. The atomic solvation parameter (ASP) for the peptide bond is comparable to the ASP of the charged carboxy terminus which is an order of magnitude larger than the ASP of the uncharged polar side chains of the Ac-X-amides. The very large peptide bond ASP, -96 +/- 6 cal/mol/A2, profoundly affects the results of computational comparisons of protein stability which use ASPs derived from octanol-water partitioning data.

Amides↗

PubMed2005

How to improve nature: study of the electrostatic properties of the surface of alpha-lactalbumin.

It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone proteins such as positively charged polyamino acids, suggesting that some fundamental aspects of the protein surface electrostatics may come into play. In the present work, the energies of charge-charge interaction in apo- and Ca(2+)-loaded alpha-lactalbumin were calculated using a Tanford-Kirkwood algorithm with either solvent accessibility correction or using a finite difference Poisson-Boltzmann method. The analysis revealed two major regions of alpha-lactalbumin that possessed highly unfavorable electrostatic potentials: (a) the Ca(2+)-binding loop and its neighboring residues and (b) the N-terminal region of the protein. Several individual mutants were prepared to neutralize specific individual surface acidic amino acids at both the N-terminus and Ca(2+)-binding loop of bovine alpha-lactalbumin. These mutants were characterized by intrinsic fluorescence, differential scanning microcalorimetry and circular dichroism. The structural and thermodynamic data agree in every case with the theoretical predictions, confirming that the N-terminal region is very sensitive to changes in charge. For example, desMet D14N mutation destabilizes protein and decreases its calcium affinity. On the other hand, desMet E1M and desMet D37N substitutions increase the thermal stability and calcium affinity. The Met E1Q is characterized by a marked increase in protein stability, whereas desMet E7Q and desMet E11L display a slight increase in calcium affinity and thermal stability. Examination of the unfavorable energy contributed by Glu1 and the energetically favorable consequences of neutralizing this residue suggests that nature may have made an error with bovine alpha-lactalbumin from the viewpoint of stabilizing structure and conformation.

Algorithms↗

PubMed2000

The 1.5 A resolution crystal structure of the carbamate kinase-like carbamoyl phosphate synthetase from the hyperthermophilic Archaeon pyrococcus furiosus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability in carbamate kinases.

Carbamoyl phosphate (CP), an essential precursor of arginine and the pyrimidine bases, is synthesized by CP synthetase (CPS) in three steps. The last step, the phosphorylation of carbamate, is also catalyzed by carbamate kinase (CK), an enzyme used by microorganisms to produce ATP from ADP and CP. Although the recently determined structures of CPS and CK show no obvious mutual similarities, a CK-like CPS reported in hyperthermophilic archaea was postulated to be a missing link in the evolution of CP biosynthesis. The 1.5 A resolution structure of this enzyme from Pyrococcus furiosus shows both a subunit topology and a homodimeric molecular organization, with a 16-stranded open beta-sheet core surrounded by alpha-helices, similar to those in CK. However, the pyrococcal enzyme exhibits many solvent-accessible ion-pairs, an extensive, strongly hydrophobic, intersubunit surface, and presents a bound ADP molecule, which does not dissociate at 22 degrees C from the enzyme. The ADP nucleotide is sequestered in a ridge formed over the C-edge of the core sheet, at the bottom of a large cavity, with the purine ring enclosed in a pocket specific for adenine. Overall, the enzyme structure is ill-suited for catalyzing the characteristic three-step reaction of CPS and supports the view that the CK-like CPS is in fact a highly thermostable and very slow (at 37 degrees C) CK that, in the extreme environment of P. furiosus, may have the new function of making, rather than using, CP. The thermostability of the enzyme may result from the extension of the hydrophobic intersubunit contacts and from the large number of exposed ion-pairs, some of which form ion-pair networks across several secondary structure elements in each enzyme subunit. The structure provides the first information on substrate binding and catalysis in CKs, and suggests that the slow rate at 37 degrees C is possibly a consequence of slow product dissociation.

Adenosine Diphosphate↗

PubMed1998

Identification of protein folding patterns using site-directed spin labeling. Structural characterization of a beta-sheet and putative substrate binding regions in the conserved domain of alpha A-crystallin.

The folding pattern of the segment of alphaA-crystallin encoded by exon 2 and containing putative substrate binding sites was explored using site-directed spin labeling (SDSL). For this purpose, a nitroxide scan was carried out between residues 60 and 108. At each site, structural constraints describing the local environment and topography were obtained from analysis of the nitroxide mobility and its solvent accessibility. Periodic patterns in the sequence-specific variation of these parameters were used to assign the secondary structure along the sequence. Geometric constraints describing the packing of secondary structure were deduced from patterns of proximities in 20 nitroxide pairs, specifically designed to differentiate between supersecondary structural motifs. Our data, in conjunction with those of Berengian et al. [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry 36, 9951-9957], reveal that the fold of the segment between residues 84 and 120 consists of an antiparallel beta-sheet of three strands arranged in consecutive beta-hairpins. The boundaries of the sheet are defined at one end by a surface of isologous association and on the other end by an unstructured, charged interdomain segment. One of the putative substrate binding segments overlaps a buried loop, suggesting that the structural origin of the thermal activation of binding is the transient exposure of this site. This paper describes and implements a general strategy for experimental fold recognition using SDSL. The results of its application to alphaA-crystallin provide the first experimental insight into the folding pattern of the subunit and establish the structural context necessary to understand molecular recognition and substrate binding.

Amino Acid Sequence↗

PubMed2005

Range and sensitivity as descriptors of molecular property spaces in dynamic QSAR analyses.

In this paper, we report the first study aimed at correlating pharmacological properties with molecular parameters derived from the physicochemical property space of bioactive molecules. A dataset of 36 ligands of the alpha(1a)-, alpha(1b)-, and alpha(1d)-adrenoceptors as published by Bremner et al. (Bioorg. Med. Chem. 2000, 8, 201-214) was used. One thousand conformers were generated for each ligand by Monte Carlo conformational analysis, and four 3D-dependent physicochemical properties were computed for each conformer of each ligand, namely virtual lipophilicity (log P), dipole moment, polar surface area (PSA), and solvent-accessible surface area (SAS). Thus, a space of four physicochemical properties was obtained for each ligand. These spaces were assessed by two descriptors, namely their range and their sensitivity (i.e., the variation amplitude of a given physicochemical property for a given variation in molecular geometric properties). Little or no correlation was found to exist between the physicochemical properties and their range or sensitivity, indicating that the latter descriptors do not encode the same molecular information as the former properties. As expected, neither the range nor the sensitivity of any of the four physicochemical properties correlated with receptor affinities. In contrast, range and sensitivity showed promising correlations with deltapK(a-b) (i.e., the alpha(1a)/alpha(1b) selectivity) for the complete dataset. The correlations were lower for deltapK(a-d) (i.e., the alpha(1a)/alpha(1d) selectivity), whereas there was no correlation at all with deltapK(b-d). These results are consistent with the results of Bremner et al., which indicate that the alpha(1a)-AR ligands bind in an extended geometry, whereas the alpha(1b)-AR and alpha(1d)-AR ligands assume more folded conformations. Since the property space descriptors presented here take structural variability into account, their correlation with deltapK(a-b) and deltapK(a-d) indicates that these selectivities are indeed driven by differences in conformational behavior and hence in property spaces.

Chemical Phenomena↗

PubMed2000

On the transferability of atomic solvation parameters: Ab initio structural prediction of cyclic heptapeptides in DMSO.

A statistical mechanics methodology for predicting the solution structures and populations of peptides developed recently is based on a novel method for optimizing implicit solvation models, which was applied initially to a cyclic hexapeptide in DMSO (C. Baysal and H. Meirovitch, Journal of American Chemical Society, 1998, vol. 120, pp. 800-812). Thus, the molecule has been described by the simplified energy function E(tot) = E(GRO) + summation operator(k) sigma(k)A(k), where E(GRO) is the GROMOS force-field energy, sigma(k) and A(k) are the atomic solvation parameter (ASP) and the solvent accessible surface area of atom k, respectively. In a more recent study, these ASPs have been found to be transferable to the cyclic pentapeptide cyclo(D-Pro(1)-Ala(2)-Ala(3)-Ala(4)-Ala(5)) in DMSO (C. Baysal and H. Meirovitch, Biopolymers, 2000, vol. 53, pp. 423-433). In the present paper, our methodology is applied to the cyclic heptapeptides axinastatin 2 [cyclo(Asn(1)-Pro(2)-Phe(3)-Val(4)-Leu(5)-Pro(6)-Val(7))] and axinastatin 3 [cyclo(Asn(1)-Pro(2)-Phe(3)-Ile(4)-Leu(5)-Pro(6)-Val(7))], in DMSO, which were studied by nmr by Mechnich et al. (Helvetica Chimica Acta, 1997, vol. 80, pp. 1338-1354). The calculations for axinastatin 2 show that special ASPs should be optimized for the partially charged side-chain atoms of Asn while the rest of the atoms take their values derived in our previous work; this suggests that similar optimization might be needed for other side chains as well. The solution structures of these peptides are obtained ab initio (i.e., without using experimental restraints) by an extensive conformational search based on E(GRO) alone and E(*)(tot), which consists of the new set of ASPs. For E(*)(tot), the theoretical values of proton-proton distances, (3)J coupling constants, and other properties are found to agree very well with the nmr results, and they are always better than those based on E(GRO).

Antineoplastic Agents↗

PubMed2001

Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI.

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.

Amides↗

PubMed1990

Ca2+ binding to calbindin D9k strongly affects backbone dynamics: measurements of exchange rates of individual amide protons using 1H NMR.

One- and two-dimensional 1H NMR have been used to study the backbone dynamics in Ca2(+)-free (apo) and Ca2(+)-loaded (Ca2) calbindin D9k at pH 7.5 and 25 degrees C. Hydrogen exchange rates of all 71 backbone amide protons (NH's) have been measured for the Ca2 form by both a direct exchange-out experiment and another experiment that measures the transfer of saturation from water protons to amide protons. A large number of NH's are found to be highly protected against exchange with solvent protons. The results for the Ca2 form are related to solvent accessibility and hydrogen bonding obtained in molecular dynamics simulations of calcium-loaded calbindin. The correlation with these parameters is strong within the N-terminal half of calbindin, which is found to be more stable than the C-terminal half. The amide proton exchange in the apo form is much faster than in the Ca2 form and was studied in a series of experiments in which the exchange was quenched after different times by Ca2+ addition. This experiment is applicable to all amide hydrogens that exchange slowly in the Ca2 form. For these NH's the effects of Ca2+ removal span from a 10(2)-fold decrease to a 10(5)-fold increase of the exchange rate, and the average is a 220-fold increase. The effects on individual NH exchange rates show that the four alpha-helices are almost intact after calcium removal and that the changes in dynamics involve not only the Ca2(+)-binding region. Hydrogen bonds involving backbone NH's in the Ca2+ loops appear to be broken or weakened when calbindin releases Ca2+, whereas the beta-sheet between the Ca2+ loops is found to be present in both the Ca2 and apo forms. Large Ca2(+)-induced effects on NH exchange rates were measured for a few residues at alpha-helix ends far from the two Ca2(+)-binding sites. This may be the result of a change in interhelix angles (or the rate of interhelix angle fluctuations) on calcium binding.

Amides↗