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At least 217 records · Page 12Linked to original sources
PubMed1998

Structural cassette mutagenesis in a de novo designed protein: proof of a novel concept for examining protein folding and stability.

The solution to the protein folding problem lies in defining the relative energetic contributions of short-range and long-range interactions. In other words, the tendency of a stretch of amino acids to adopt a final secondary structural fold is context dependent. Our approach to this problem is to address whether an amino acid sequence, a "cassette," with a defined secondary structure in the three-dimensional structure of a native protein, can adopt a different conformation when placed into a different protein environment. Thus, we designed de novo a disulfide-bridged two-stranded alpha-helical parallel coiled coil, where each polypeptide chain consisted of 39 residues, as a "cassette holder." The 11-residue cassette would be inserted into the center of each polypeptide chain between the two nucleating alpha-helices to replace the control sequence. This Structural Cassette Mutagenesis model permits the analysis of short-range interactions within the inserted cassette as well as long-range interactions between the nucleating helices and the cassette region. The cassette holder, with a control sequence as the cassette, had a GdnHCl transition midpoint during denaturation of 5.6M. To demonstrate the feasibility of our model, an 11-residue beta-strand cassette from an immunoglobulin fold was inserted. The cassette was fully induced into the alpha-helical conformation with a [GdnHCl]1/2 value of 3.2M. To demonstrate the importance of short-range interactions (beta-sheet/alpha-helical propensities of amino acid side chains) in modulating structure and stability, a series of 1-5 threonine residues (highest beta-sheet propensity) were substituted into the solvent-exposed portions of the cassette in the alpha-helical conformation. Each successive substitution systematically decreased the stability of the coiled coil with peptide T4b (4 Thr residues) having a [GdnHCl]1/2 value of 2.2M. The single substitution of Ile in the hydrophobic core of the cassette with Ala or Thr had the most dramatic effect on protein stability (peptide 120T, [GdnHCl]1/2 value of 1.4M). Though these substitutions were able to modulate stability, they were not able to disrupt the alpha-helical conformation of the cassette, showing the importance of the nucleating alpha-helices on either side of the cassette in controlling conformation of the cassette. We have demonstrated the feasibility of our model protein to accept a beta-strand cassette. The effect of cassettes containing other beta-strands, beta-turns, loops, regions of undefined structure, and helical segments on conformation and stability of our model protein will also be determined.

Amino Acid Sequence↗
PubMed1993

Solvent effects on chemical processes. V: Hydrophobic and solvation effects on the solubilities of substituted biphenyls in methanol/water mixtures.

A phenomenological model that permits solvent effects to be separated into general medium effects (the solvophobic effect) and solvation effects is applied to the solubility of a series of biphenyl compounds in methanol/water mixtures. The parameters of the model (gA, K1, and K2, K1 and K2 are equilibrium constants for solvation and gA describes the general medium contribution) were evaluated from the nonlinear regression of the model equation to the data. It was found that the surface tension curvature factor (g) was 0.37, that A represents the hydrophobic (nonpolar) surface area of the solute molecule, and that K1 and K2 were 2.53 and 1.77 (means for solutes in methanol/water), respectively. These results permit solvent effects on solubility in methanol/water to be predicted and they refine the interpretation of the solvent effect model.

Biphenyl Compounds↗
PubMed1999

Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix.

Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala --> Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol. 6, 210-212]. These residues also show similar changes in DeltaG(HX) upon Ala --> Gly mutations (DeltaDeltaG(HX)) as compared to equilibrium measurements (DeltaDeltaG(U)), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3833-2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala --> Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.

Alanine↗
PubMed1976

Solvent-dependent conformational system in hydroxyureas detected by NMR spectroscopy.

The proton magnetic resonance spectra of the antileukemia agent hydroxyurea and substituted hydroxyureas in several solvents were recorded and correlated with structural features. Solvent-dependent differences in conformational preferences due to effects on internal hydrogen bonding, a temperature-dependent conformational feature, anthe exchangeability of protons with deuterium oxide and acetone-d6 were observed. The conformational features consistent with the spectral data are discussed.

Chemical Phenomena↗
PubMed1998

Soluble polymer traceless linker investigations: solvent effects on the desulfonylation of polyethylene glycol (PEG) substituted aryl alkyl sulfones with sodium amalgam.

The soluble polymer polyethylene glycol containing a traceless aryl alkyl sulfone linker was found to be resistant to reductive cleavage with sodium amalgam (Na/Hg). Detailed investigations revealed that the solvent plays an important role in the desulfonylation reaction; subsequently it was determined that MeOH/DMF (1/8) is an ideal solvent system for the cleavage of this traceless linker.

Alloys↗
PubMed1994

Use of amino acid environment-dependent substitution tables and conformational propensities in structure prediction from aligned sequences of homologous proteins. I. Solvent accessibility classes.

Buried and exposed residues are predicted by composing amino acid substitution patterns and mean propensities for the two solvent accessibility classes with the amino acid residues at equivalent sites in aligned sequences of homologous proteins. In a study of 13 protein families, the accuracy of the prediction is around 77% (the correlation coefficient between the predicted and observed accessibility classes is 0.52). The environment dependent amino acid substitution tables are especially important in prediction of buried hydrophilic and exposed hydrophobic residues, which are not well predicted with propensities alone. Since the prediction is site-specific in the sense that any averaged properties over neighbouring residues are not required, the results can be used for the prediction of secondary structures by detecting periodicity in the sequence of buried and exposed classes.

Amino Acid Sequence↗
PubMed1994

In vivo characterization of mutants of the bacteriophage f1 gene V protein isolated by saturation mutagenesis.

The gene V protein of bacteriophage f1 binds to single-stranded nucleic acids and is essential for propagation of phage f1. We tested the function of gene V protein mutants with single amino acid substitutions in two ways: by the ability of the mutant proteins to support phage growth, and by the ability of the mutant proteins, when expressed at high levels, to inhibit the growth of Escherichia coli. The results of the tests were used to identify sites in the protein that are relatively tolerant or intolerant to substitution, where tolerant sites are defined as those where most substitutions do not affect the function of the protein. The two assays generally yielded similar results for the tolerance of sites to substitution. Many sites that are less than 10% exposed to the solvent are relatively intolerant of substitution, with even very conservative substitutions leading to loss of function in some cases such as Ile to Leu at residue 6. Some buried sites such as Ile47 are more tolerant, with even a substitution of Ile to Thr leading to a functional protein based on the ability of the proteins to inhibit the growth of E. coli. Some surface sites in the protein (> 10% exposure to solvent) that are thought to be near the location of bound oligonucleotides, such as Arg16, Val19, Ser20, Arg21, Tyr26, Lys46 and Arg80, are sensitive to substitution. Other side-chains thought to be close to bound oligonucleotides, including Leu28, can be replaced with a number of amino acids with little loss of function based on either assay. Most non-Gly/Pro surface residues thought to be distant from the locations of bound oligonucleotides are relatively tolerant of substitution, except for two small residues (Ala11 and Thr14), two aromatic residues (Tyr34 and Tyr56), two residues that are only partially exposed to solvent (Asn29 and Val70), and three residues that have been proposed to be at the dimer-dimer interface formed when gene V protein binds to nucleic acids (Glu40, Tyr41 and Arg82).

DNA, Single-Stranded↗
PubMed2006

Dynamic thermodynamic resolution: solvent effects, mechanism, and an asymmetric 3,4,5-substituted benzazepine synthesis.

[reaction: see text] The resolution in the lithiation-substitution sequence from 1 to 4-11 in MTBE is shown to be under thermodynamic control in contrast to the previous report of kinetic control in diethyl ether. Diastereomeric equilibration of a soluble complex is shown to be controlling and an asymmetric synthesis of a 3,4,5-substituted benzazepine is reported.

Benzazepines↗
PubMed1980

Comparison of theoretical and experimental approaches to determination of conformation of nucleosides about the glycosidic bond.

A study has been made by means of 1H-NMR spectroscopy of the syn in equilibrium anti dynamic equilibrium about the glycosidic bond for 5'-deoxyadenosine and some 8-substituted analogues, in different solvents. The results are compared with those previously obtained for the parent adenosine and its 8-substituted analogues. Quantum chemical calculations, with the aid of the Classical Potential and PCILO procedures, were applied to obtain the energies for different conformations of the base in adenosine and 5'-deoxyadenosine, and their 8-methyl and 8-halogeno derivatives. Good agreement was found between experimentally determined conformations in solution and those corresponding theoretically to the energy minima, particularly those calculated by the PCILO method. Comparison of the quantitative experimental data with the theoretical results was used to evaluate the validity of the latter and their applicability to studies of nucleoside conformation. The experimental and theoretical findings pointed to the existence of a marked flexibility about the glycosidic bond of the parent nucleosides and their 8-substituted analogues, when the 8-substituents were not too bulky, such as methyl or bromine. Considerations is given to possible correlations between conformational parameters in nucleosides and their 5'-deoxy analogues. It is shown that the proposed stabilization of the conformation syn by intramolecular hydrogen bonding, 5'-OH...N(3), is not in accord with the results of the present study.

Adenosine↗
PubMed2005

Solvent induced different morphologies of bis(propyl)triethoxysilane substituted perylenediimide and their optical properties.

Nano/micro-structure of bis(propyl)triethoxysilane substituted perylenediimide (1) with nanoparticle and twisted microrod morphologies were obtained by reprecipitation method induced by water and petroleum ether, respectively. It is believed that the different nucleation and growth processes involved are responsible for the formation of the nano/micro-structure with different morphologies of 1. UV-vis absorption and photoluminescence measurements show that their UV-vis absorption and photoluminescence properties are different from each other as well as their monomer and bulk materials due to the different effects on the charge transfer (CT) transition energy levels caused by their different aggregation behaviors.

Journal Article↗
PubMed2006

Synthetic and structural comparisons of bismuth(III) carboxylates synthesised under solvent-free and reflux conditions.

Two synthetic approaches to the formation of bismuth(III) carboxylates have been explored and compared. Ph(3)Bi was reacted with a series of carboxylic acids (RCO(2)H) of varying pK(a) and functionality (R = PhCH[double bond, length as m-dash]CH, o-MeOC(6)H(4), m-MeOC(6)H(4), o-H(2)NC(6)H, o-O(2)NC(6)H(4), p-O(2)NC(6)H(4), 2-(C(5)H(4)N)) under reflux conditions in toluene and solvent-free. The thermochemical profiles of the solvent-free reactions were also studied by DSC-TGA. All reactions produced the tri-substituted bismuth carboxylates in comparable yields and purity with the exceptions of picolinic acid and p-nitrobenzoic acid. 2-Picolinic acid exclusively formed the di-substituted complex, [PhBi(2-(C(5)H(4)N)CO(2))(2)](4), by both methods, while p-nitrobenzoic acid gave the tri-substituted complex through reflux and the di-substituted complex under solvent-free conditions. Two of the complexes were structurally authenticated by single crystal X-ray diffraction: [PhBi(2-(C(5)H(4)N)CO(2))(2)](4) is tetrameric formed through five membered chelate rings involving the pyridyl N and O(-C) rather than the less stable carboxylate (-CO(2)) chelates, while [Bi(o-MeOC(6)H(4)CO(2))(3)](infinity), is a polymer in which dimeric units, constructed around two chelating and one unsymmetrical bridging carboxylate on each Bi centre, are then joined together through longer intermolecular Bi-O bridging bonds.

Journal Article↗
PubMed2005

Solvent control on the selective, nonselective, and absent response of a partially substituted lower rim calix(4)arene derivative for soft metal cations (mercury(II) and silver(I)). Structural and thermodynamic studies.

The solvent control on the ability of a partially substituted lower rim calix(4)arene derivative 5,11,17,23,tetra-tert-butyl[25,27-bis(hydroxy)-26,28-bis(ethylthioethoxy)]-calix(4)arene, 1 to host soft metal cations (Hg(II) and Ag(I)) is demonstrated through 1H NMR, electrochemical (conductance measurements), and thermodynamic characterization of the complexation process in a wide variety of solvents. Solvent-ligand interactions were assessed from 1H NMR measurements involving 1 and various solvents in CDCl3. Thus, the formation of a 1:1 1-CH3CN adduct is reported. As far as metal cations are concerned, depending on the medium their complexation with 1 was only observed for Hg(II) and Ag(I). Thus, in acetonitrile, 1 is more selective for Hg(II) relative to Ag(I) by a factor of 2.2 x 10(3). In methanol the selectivity is reversed to an extent that the affinity of 1 for Ag(I) is 1.4 x 10(3) higher than that for Hg(II). However, 1 is unable to recognize selectively these cations in N,N-dimethylformamide while in propylene carbonate the ability of 1 to interact with these cations is lost. An outstanding feature of thermodynamics emerges when an assessment is made of the ligand effect on the complexation of these cations and analogues calix(4)arene derivatives. Thus, in acetonitrile the thermodynamics of cation complexation by the hydrophilic cavity of a calix(4)arene containing mixed pendant groups is built up from thermodynamic data for the same process involving derivatives with common functionalities at the narrow rim. This is a unique example of the additive contribution of pendant arms in the field of thermodynamics of calixarene chemistry.

Acetonitriles↗
PubMed1998

Mechanisms of spectral tuning in blue cone visual pigments. Visible and raman spectroscopy of blue-shifted rhodopsin mutants.

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. We identified 12 amino acid residues in the human blue cone pigment that might induce the required green-to-blue opsin shift. The simultaneous substitution of nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nm, accounting for 2,830 cm-1, or 80%, of the opsin shift between rhodopsin and the blue cone pigment. Raman spectroscopy of mutant pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered. An increase in the number of dipolar side chains near the protonated Schiff base of retinal increases the ground-excited state energy gap via long range dipole-dipole Coulomb interaction. In addition, the W265Y substitution causes a decrease in solvent polarizability near the chromophore ring structure. Finally, two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments.

Amino Acid Sequence↗
PubMed1987

Cyclic nucleotide binding to cAMP receptor protein from Escherichia coli. Optical and ligand-binding studies.

cAMP receptor protein from Escherichia coli has been purified on a large scale. Analogues of cAMP modified on the 6-NH2 group of the adenosine ring, the ribose 2'OH group or the cyclic phosphate are able to displace cAMP from its binding site with dissociation constants of similar magnitude to that of cAMP. More extensive modification produces weaker binding. Ultraviolet/visible difference spectroscopy and fluorescence spectroscopy show that the environment of the bound adenosine moiety is considerably less polar than that in aqueous solvent, while an anthraniloyl group substituted on the 2'OH position remains accessible to solvent. The 2-NH2 group of cGMP appears to be protonated in the bound form, while no change in the charge state of cAMP is apparent.

Bacterial Proteins↗
PubMed1990

Protein stability and electrostatic interactions between solvent exposed charged side chains.

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.

Apoproteins↗