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Showing papers in "Proteins in 1986"


Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: In this article, the authors reported the implementation of a finite-difference algorithm which solves the linearized Poisson-Boltzmann equation for molecules of arbitrary shape and charge distribution, including the screening effects of electrolytes.
Abstract: In this paper we report the implementation of a finite-difference algorithm which solves the linearized Poisson-Boltzmann equation for molecules of arbitrary shape and charge distribution and which includes the screening effects of electrolytes. The microcoding of the algorithm on an ST-100 array processor allows us to obtain electrostatic potential maps in and around a protein, including the effects of ionic strength, in about 30 minutes. We have applied the algorithm to a dimer of the protein Cu-Zn superoxide dismutase (SOD) and compared our results to those obtained from uniform dielectric models based on coulombic potentials. We find that both the shape of the protein-solvent boundary and the ionic strength of the solvent have a profound effect on the potentials in the solvent. For the case of SOD, the cluster of positive charge at the bottom of the active site channel produces a strongly enhanced positive potential due to the focusing of field lines in the channel—a result that cannot be obtained with any uniform dielectric model. The remainder of the protein is surrounded by a weak negative potential. The electrostatic potential of the enzyme seems designed to provide a large cross-sectional area for productive collisions. Based on the ionic strength dependence of the size of the positive potential region emanating from the active site and the repulsive negative potential barrier surrounding the protein, we are able to suggest an explanation for the ionic strength dependence of the activity of the native and chemically modified forms of the enzyme.

704 citations


Journal ArticleDOI
01 Mar 1986-Proteins
TL;DR: The properties of the TFE stabilization indicate that TFE might be a useful probe with which to measure the stability of marginally stable peptides and small proteins.
Abstract: The effects of trifluoroethanol (TFE) on the stability of the alpha-helix formed by ribonuclease S-peptide, residues 1-19 of ribonuclease A, were studied by measuring circular dichroism as a function of TFE concentration, pH, and temperature. The S-peptide forms an unusually stable alpha-helix, which is known to be stabilized by TFE. The magnitude of the effect of charged groups on the peptide, manifested by the change in alpha-helix stability as a function of pH, was not altered significantly by either TFE concentration or temperature, indicating that the lower dielectric constant of TFE is not important in the stabilization of this alpha-helix. This suggests that the alpha-helix might be stabilized by many interactions in addition to the effects of charges. The titration curve of circular dichroism vs. TFE concentration appears to be cooperative at 0 degree C, but becomes progressively less cooperative at temperatures between 25 and 75 degrees C. The properties of the TFE stabilization indicate that TFE might be a useful probe with which to measure the stability of marginally stable peptides and small proteins.

349 citations


Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction.
Abstract: Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.

259 citations


Journal ArticleDOI
01 Feb 1986-Proteins
TL;DR: The feasibility of determining the conformation of segments of polypeptide chain up to six residues in length in globular proteins by means of systematic search through the possibles conformations has been investigated.
Abstract: The feasibility of determining the conformation of segments of a polypeptide chain up to six residues in length in globular proteins by means of a systematic search through the possible conformations has been investigated. Trial conformations are generated by using representative sets of phi, psi, and chi angles that have been derived from an examination of the distributions of these angles in refined protein structures. A set of filters based on simple rules that protein structures obey is used to reduce the number of conformations to a manageable total. The most important filters are the maintenance of chain integrity and the avoidance of too-short van der Waals contacts with the rest of the protein and with other portions of the segment under construction. The procedure is intended to be used with approximate models so that allowance is made throughout for errors in the rest of the structure. All possible main chains are first constructed and then all possible side-chain conformations are built onto each of these. The electrostatic energy, including a solvent screening term, and the exposed hydrophobic area are evaluated for each accepted conformation. The method has been tested on two segments of chain in the trypsin like enzyme from Streptomyces griseus. It is found that there is a wide spread of energies among the accepted conformations, and the lowest energy ones have satisfactorily small root mean square deviations from the X-ray structure.

249 citations


Journal ArticleDOI
01 Apr 1986-Proteins
TL;DR: A method for predicting the conformations of loops in proteins and its application to four of the complementarity determining regions in the crystallographically determined structure of MCPC603 suggests that these loops fold independently of sequence variation.
Abstract: We describe a method for predicting the conformations of loops in proteins and its application to four of the complementarity determining regions [CDRs] in the crystallographically determined structure of MCPC603. The method is based on the generation of a large number of randomly generated conformations for the backbone of the loop being studied, followed by either minimization or molecular dynamics followed by minimization starting from these random structures. The details of the algorithm for the generation of the loops are presented in the first paper in this series (Shenkin et al. [submitted]). The results of minimization and molecular dynamics applied to these loops is presented here. For the two shortest CDRs studied (H1 and L2, which are five and seven amino acids long), minimizations and dynamics simulations which ignore interactions of the loop amino acids beyond the carbon beta replicate the conformation of the crystal structure closely. This suggests that these loops fold independently of sequence variation. For the third CDR (L3, which is nine amino acids), those portions of the CDR near its base which are hydrogen bonded to framework are well replicated by our procedures, but the top of the loop shows significant conformational variability. This variability persists when side chain interactions for the MCPC603 sequence are included. For a fourth CDR (H3, which is 11 amino acids long), new low-energy backbone conformations are found; however, only those which are close to the crystal are compatible with the sequence when side chain interactions are taken into account. Results from minimization and dynamics on single CDRs with all other CDRs removed are presented. These allow us to explore the extent to which individual CDR conformations are determined by interactions with framework only.

201 citations


Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: The crystal structure of the Fab of the galactan‐binding immunoglobulin J539 (a mouse IgA,κ) has been determined at a resolution of approximately 2.6 Å by X‐ray diffraction using the starting model obtained from the real space search described previously.
Abstract: The crystal structure of the Fab of the galactan-binding immunoglobulin J539 (a mouse IgA,kappa) has been determined at a resolution of approximately 2.6 A by X-ray diffraction. The starting model was that obtained from the real space search described previously (Navia, M.A., Segal, D.M., Padlan, E.A., Davies, D.R., Rao, D.N., Rudikoff, S. and Potter, M. "Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5 A resolution." Proc. Nat. Acad. Sci. USA, 76:4071-4074, 1979). This Fab structure has now been refined by restrained least-squares procedures to an R-value of 19% for the 11,690 unique reflections between 8.0 A and 2.6 A. The rms deviation from ideal bond lengths is 0.025 A. The overall structure differs from McPC603 Fab, another mouse IgA,kappa antibody, in that the elbow bend, relating the variable and constant parts of the molecule, is 145 degrees vs. 133 degrees for McPC603. The region of the molecule expected to be the antigen binding site contains a large cavity with two clefts leading away from it. This has been fitted with a model of an oligo-galactan.

162 citations


Journal ArticleDOI
01 Apr 1986-Proteins
TL;DR: The refined, 1.8‐Å crystal structures of the wild‐type and 7150 subtilisin have been compared in detail, leading to the conclusion that slight improvements in hydrogen bond parameters in the vicinity of position 218 result in the enhanced thermal stability of 7150.
Abstract: A procedure has been developed for the isolation and identification of mutants in the bacterial serine protease subtilisin that exhibit enhanced thermal stability. The cloned subtilisin BPN' gene from Bacillus amyloliquefaciens was treated with bisulfite, a chemical mutagen that deaminates cytosine to uracil in single-stranded DNA. Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermal stability were selected by a simple plate assay procedure which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures. One thermostable subtilisin variant, designated 7150, has been fully characterized and found to differ from wild-type subtilisin by a single substitution of Ser for Asn at position 218. The 7150 enzyme was found to undergo thermal inactivation at one-fourth the rate of the wild-type enzyme when incubated at elevated temperatures. Moreover, the mid-point in the thermally induced transition from the folded to unfolded state was found to be 2.4-3.9 degrees C higher for 7150 as determined by differential scanning calorimetry under a variety of conditions. The refined, 1.8-A crystal structures of the wild-type and 7150 subtilisin have been compared in detail, leading to the conclusion that slight improvements in hydrogen bond parameters in the vicinity of position 218 result in the enhanced thermal stability of 7150.

148 citations


Journal ArticleDOI
01 Apr 1986-Proteins
TL;DR: The sequence of the H subunit is reported, completing the primary structure determination of the reaction center from R. sphaeroides and showing that the L and M subunits of reaction centers and D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence.
Abstract: The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.

144 citations


Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: Studies showing that the addition of γ subunit also reverses the activation of PDE by Tα‐GTP‐γS are reported, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of Tα-GTP.
Abstract: The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.

140 citations



Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: Twelve‐ and sixteen‐residue peptides have been designed to form tetrameric alphahelical bundles, and the design can be extended to a four‐alpha‐helical bundle formed from a single polypeptide by adding three peptide linkers.
Abstract: Twelve- and sixteen-residue peptides have been designed to form tetrameric alpha-helical bundles. Both peptides are capable of folding into amphiphilic alpha-helices, with leucyl residues along one face and glutamyl and lysyl residues along the opposite face. Four such amphiphilic alpha-helices are capable of forming a noncovalently bonded tetramer. Neighboring helices run in antiparallel directions in the design, so that the complex has 222 symmetry. In the designed tetramer, the leucyl side chains interdigitate in the center in a hydrophobic interaction, and charged side chains are exposed to the solvent. The designed 12-mer (ALPHA-1) has been synthesized, and it forms helical aggregates in aqueous solution as judged by circular dichroic spectroscopy. It has also been crystallized and characterized by x-ray diffraction. The crystal symmetry is compatible with (but does not prove) the design. The design can be extended to a four-alpha-helical bundle formed from a single polypeptide by adding three peptide linkers.

Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: The basic pancreatic trypsin inhibitor was applied by using rapid mixing techniques and 1H NMR to provide site‐resolved kinetic information on the early phases of protein structure acquisition.
Abstract: A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin, which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and /sup 1/H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion By using /sup 1/H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained For eight individual amide protons located in the beta-sheetmore » and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed These rates are on the time scale of the fast folding phase observed with optical probes« less

Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: Oligonucleotide‐directed mutagenesis has been used to replace α‐helical glycines in the N‐terminal domain of λ repressor with alanines, and it is shown that the Gly46→Ala substitution, the Gly48→AlA substitution, and the double substitution increase the melting temperature of the N-terminaldomain by 3–6°.
Abstract: Oligonucleotide-directed mutagenesis has been used to replace alpha-helical glycines in the N-terminal domain of lambda repressor with alanines. Since alanine is a significantly better helix-forming residue than glycine, these changes were predicted to have a stabilizing effect. We show that the Gly46----Ala substitution, the Gly48----Ala substitution, and the double substitution increase the melting temperature of the N-terminal domain by 3-6 degrees.

Journal ArticleDOI
01 Feb 1986-Proteins
TL;DR: The GTP‐binding subunit of transducin (Tα) activates the cGMP phosphodiesterase (PDE) of bovine retinal rods by relieving the constraint imposed by the inhibitory subunit PDEγ, and the complex Tα.GTPγS‐PDEγ formed when Tα is activated by the nonhydrolyzable analog G TPγS.
Abstract: The GTP-binding subunit of transducin (T alpha) activates the cGMP phosphodiesterase (PDE) of bovine retinal rods by relieving the constraint imposed by the inhibitory subunit PDE gamma. We have isolated and characterized the complex T alpha.GTP gamma S-PDE gamma formed when T alpha is activated by the nonhydrolyzable analog GTP gamma S. Sedimentation and light-scattering techniques demonstrate that, in contrast to free T alpha.GTP gamma S, which is soluble, the T alpha.GTP gamma S-PDE gamma complex, as well as T alpha.GTP-PDE gamma, is membrane bound at cytosolic ionic strength. It is eluted from the membrane at low ionic strength as a monomeric and 1:1 stoichiometric complex. The relative affinities of PDE gamma for PDE alpha beta and for T alpha.GTP are discussed.

Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: This work has asked whether, in semisynthetic ribonuclease S reconstituted from S‐protein plus an analogue of S‐peptide (1–15), the stability of the peptide helix is correlated with the Tm of the reconst ituted ribonuclelease S.
Abstract: Recent work has shown that, with synthetic analogues of C-peptide (residues 1-13 of ribonuclease A), the stability of the peptide helix in H2O depends strongly on the charge on the N-terminal residue. We have asked whether, in semisynthetic ribonuclease S reconstituted from S-protein plus an analogue of S-peptide (1-15), the stability of the peptide helix is correlated with the Tm of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9----Leu, a blocked alpha-COO- group (-CONH2), and either Gln11 or Glu11. The N-terminal residue has been varied; its charge varies from +2 (Lys) to -1 (succinyl-Ala). We have measured the stability of the peptide helix, the affinity of the peptide for S-protein (by C.D. titration), and the thermal stability of the reconstituted ribonuclease S. All six peptide analogues show strongly enhanced helix formation compared to either S-peptide (1-15) or (1-19), and the helix content increases as the charge on the N-terminal residue changes from +2 to -1. All six peptides show increased affinity for S-protein compared to S-peptide (1-19), and all six reconstituted ribonucleases S show an increase in Tm compared to the protein with S-peptide (1-19). The Tm increases as the charge on residue 1 changes from +2 to -1. The largest increment in Tm is 6 degrees. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure.

Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: It is found that the C‐terminal domain has polymerase activity but no measurable 3′‐5′ exonuclease activity, consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.
Abstract: The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Journal ArticleDOI
01 Feb 1986-Proteins
TL;DR: Application of methods described herein, combined with appropriate scale‐up fermentation capabilities, provides the means for generating gram quantities of human renin and its zymogen.
Abstract: Transfection of Chinese hamster ovary (CHO) cells with a plasmid containing the cDNA for human preprorenin has provided cell lines that secrete 15 mg of native prorenin per liter of culture medium. Tryptic activation of the prorenin occurs by selective cleavage of the Arg66-Leu67 bond (numbering as in preprorenin). The renin product, purified in a single step and in high yield by affinity chromatography, is fully stable for as long as 8 months when stored in solution at 4°C and pH 6.5. Purity of the renin was judged to be greater than 95% by gel electrophoresis, compositional and N-terminal sequence analyses, and specific enzyme activity. An important aspect of the present work is the development of a direct assay for renin which permits accurate and reproducible evaluation of enzyme units and kinetic parameters. Application of methods described herein, combined with appropriate scale-up fermentation capabilities, provides the means for generating gram quantities of human renin and its zymogen.

Journal ArticleDOI
01 Mar 1986-Proteins
TL;DR: The N‐terminal amino acid sequence and amino acid composition demonstrated that mcbA is the structural gene for microcin B17, a low‐molecular‐weight protein that inhibits DNA replication in a number of enteric bacteria.
Abstract: Microcin B17 is a low-molecular-weight protein that inhibits DNA replication in a number of enteric bacteria. It is produced by bacterial strains which harbor a 70-kilobase plasmid called pMccB17. Four plasmid genes (named mcbABCD) are required for its production. The product of the mcbA gene was identified by labelling minicells. The mcbA gene product was slightly larger when a mutation in any of the other three production genes was present. This indicates that these genes are involved in processing the primary mcbA product to yield the active molecule. The mcbA gene product predicted from the nucleotide sequence has 69 amino acids including 28 glycine residues. Microcin B17 was extracted from the cells by boiling in 100 mM acetic acid, 1 mM EDTA, and purified to homogeneity in a single step by high-performance liquid chromatography through a C18 column. The N-terminal amino acid sequence and amino acid composition demonstrated that mcbA is the structural gene for microcin B17. The active molecule is a processed product lacking the first 26 N-terminal residues. The 43 remaining residues include 26 glycines. While microcin B17 is an exported protein, the cleaved N-terminal peptide does not have the characteristic properties of a "signal sequence", which suggests that it is secreted by a mechanism different from that used by most secreted proteins of E. coli.

Journal ArticleDOI
01 Apr 1986-Proteins
TL;DR: Polypeptide structural predictions suggest that the HSV‐1 N‐terminal domain may be separated into two regions, namely, a β‐sheet structure followed by a nonstructured area and blocks of amino acids conserved between the different ribonucleotide reductases, which may be important for enzyme activity.
Abstract: Herpes simplex virus type 1 (HSV-1) encodes a ribonucleotide reductase which comprises two polypeptides with sizes of 136,000 (RR1) and 38,000 mol. wt. (RR2). We have determined the entire DNA sequence specifying HSV-1 RR1 and have identified two adjacent open reading frames in varicella-zoster virus (VZV) which have homology to HSV RR1 and RR2; the predicted sizes for the VZV RR1 and RR2 polypeptides are 87,000 and 35,000 mol. wt. respectively. Amino acid comparisons with RR1 and RR2 polypeptides from other organisms indicate that HSV-1 RR1 contains a unique N-terminal domain which is absent from other RR1 polypeptides apart from HSV-2 RR1. These N-terminal amino acid sequences are poorly conserved between HSV-1 and HSV-2 in contrast to the remainder of the protein which shows greater than 90% homology. Polypeptide structural predictions suggest that the HSV-1 N-terminal domain may be separated into two regions, namely, a beta-sheet structure followed by a nonstructured area. Across the remainder of RR1 and RR2, comparisons also reveal blocks of amino acids conserved between the different ribonucleotide reductases, and these may be important for enzyme activity. From predictions on the structure of these conserved blocks, we have proposed that the location of a substrate binding site within RR1 is centered on three conserved glycine residues in a region which is predicted to adopt a beta-sheet/turn/alpha-helical structure; this approximates to the structure for ADP nucleotide binding folds. Finally, we propose that the promoters for the HSV and Epstein-Barr virus (EBV) RR2 transcripts have evolved by separate evolutionary routes.

Journal ArticleDOI
01 Feb 1986-Proteins
TL;DR: Results suggest that when quenching is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface, and available crystallographic results support this conclusion.
Abstract: Experiments were done to test the thesis that acrylamide and similar small molecules can penetrate into proteins on a nanosecond time scale. The approach taken was to measure the pat- tern of fluorescence quenching exhibited by quench- ing molecules differing in molecular character (size, polarity, charge) when these are directed against protein tryptophans that cover the whole range of tryptophan accessibility. If quenching involves pro- tein penetration and internal quencher migration, one expects that larger quenchers and more polar quenchers should display lesser quenching. In fact, no significant dependence on quencher character was found. For proteins that display measurable quench- ing, the disparate quenchers studied display very similar quenching rate constants when directed against any particular protein tryptophan. For sev- eral proteins having tryptophans known to be buried, no quenching occurs. These results are not consis- tent with the view that the kinds of small molecules studied can quite generally penetrate into and dif- fuse about within proteins at near-diffusion-limited rates. Rather the results suggest that when quench- ing is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface. Available crystallographic results support this conclusion.

Journal ArticleDOI
01 Apr 1986-Proteins
TL;DR: It is argued that these N‐terminal residues are important for operator recognition but that they are not part of a conventional helix‐turn‐helix DNA binding structure and that Arc may use a new mechanism for sequence specific DNA binding.
Abstract: We have isolated 64 different missense mutations at 36 out of 53 residue positions in the Arc repressor of bacteriophage P22. Many of the mutant proteins with substitutions in the C-terminal 40 residues of Arc have reduced intracellular levels and probably have altered structures or stabilities. Mutations in the N-terminal ten residues of Arc cause large decreases in operator DNA binding affinity without affecting the ability of Arc to fold into a stable three-dimensional structure. We argue that these N-terminal residues are important for operator recognition but that they are not part of a conventional helix-turn-helix DNA binding structure. These results suggest that Arc may use a new mechanism for sequence specific DNA binding.


Journal ArticleDOI
01 Mar 1986-Proteins
TL;DR: CPP is shown to give better results than other commonly used modeling procedures when applied to the same problem and to be accurate to the extent that both of the sequence differences in the region are modeled correctly and that conformational changes in a number of nearby residues are correctly identified.
Abstract: A procedure (coupled perturbation procedure, CPP) is introduced as a specific method for calculating the detailed three-dimensional structure of a protein molecule which has a number of amino-acid substitutions relative to some previously determined "parent" protein structure. The accuracy of the procedure is tested by calculating the conformation of a region of the human immunoglobulin fragment Fab Kol based on the analogous region of the human immunoglobulin fragment Fab New. Both structures have previously been determined crystallographically. The calculated model is accurate to the extent that both of the sequence differences in the region are modeled correctly and that conformational changes in a number of nearby residues are correctly identified. CPP is shown to give better results than other commonly used modeling procedures when applied to the same problem.

Journal ArticleDOI
01 Mar 1986-Proteins
TL;DR: NaDodSO4‐polyacrylamide gel electrophoresis of [3H]retinal‐labeled membrane proteins from the mutants indicates SR and the 480‐nm pigment contain distinct chromophoric polypeptides differing in their migration rates.
Abstract: A second slow-cycling retinylidene protein, in addition to slow-cycling (sensory) rhodopsin (SR), can be bleached with hydroxylamine and regenerated with all-trans retinal in photosensory signaling Halobacterium halobium membranes. Flash photolysis shows this protein undergoes a photochemical reaction cycle characterized by photoconversion of its ground state (lambda max 480 nm) to a species with lambda max less than or equal to 360 nm, which thermally regenerates the 480-nm species with a t1/2 of 260 msec at 25 degrees C, under conditions in which SR photocycles at 650 msec in the same membranes. Mutants characterized with respect to their phototaxis behavior are identified which contain SR and the 480-nm pigment, the latter ranging from undetectable to a concentration equal to that of SR. Receptor mutants lacking all phototaxis sensitivity lack both of the photochemically reactive proteins. The mutant properties contribute to an accumulation of behavioral and spectroscopic evidence that the 480-nm pigment is a second sensory photoreceptor in H. halobium. NaDodSO4-polyacrylamide gel electrophoresis of (/sup 3/H)retinal-labeled membrane proteins from the mutants indicates SR and the 480-nm pigment contain distinct chromophoric polypeptides differing in their migration rates. The data implicate polypeptides of 25,000 Mr and 23,000 Mr as retinal-binding polypeptides of SR andmore » the 480-nm protein, respectively.« less

Journal ArticleDOI
01 Apr 1986-Proteins
TL;DR: The genes for the two enzymes appear to have arisen by shuffling of two conserved sequences and either one or two other sequences.
Abstract: Cellulomonas fimi produces an endoglucanase and an exoglucanase which bind strongly to cellulose. Each enzyme contains three distinct regions: a short sequence of about 20 amino acids containing only proline and threonine (the Pro-Thr box); an irregular region, rich in hydroxyamino acids, of low charge density, and which is predicted to have little secondary structure; and an ordered region of higher charge density which contains a potential active site, and which is predicted to have secondary structure. The Pro-Thr box is conserved almost perfectly in the two enzymes. The irregular regions are 50% conserved, and the conserved sequences include four Asn-Xaa-Ser/Thr sites. The ordered regions appear not to be conserved, but the potential active sites both have the sequence Glu-Xaa7-Asn-Xaa6-Thr; they occur at widely separated sites in the two regions. The order of the regions is reversed in the two enzymes: irregular-Pro-Thr box-ordered in the endoglucanase; ordered-Pro-Thr box-irregular in the exoglucanase. The genes for the two enzymes appear to have arisen by shuffling of two conserved sequences and either one or two other sequences.

Journal ArticleDOI
01 Mar 1986-Proteins
TL;DR: Nine mutants were made along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site, and eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule.
Abstract: Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr-cleaved C-terminal fragments with channel-forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2-terminus of the channel-forming domain of colicin E1 appears therefore to lie between residues 428 and 447. Since, however, the last six C-terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule. It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six alpha-helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.

Journal ArticleDOI
01 Apr 1986-Proteins
TL;DR: In order to characterize the zymogen of the milk‐clotting enzyme from Rhizomucor miehei, a cDNA library on pBR327 in Escherichia coli was constructed and the cDNA sequence corresponding to a precursor form of the enzyme was determined.
Abstract: In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The decuced amino sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acide residues with a total molecular weight of 38,701. Clusters of idendtities around the active site cleft support the assumption that these proteinasess have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.

Journal ArticleDOI
01 Mar 1986-Proteins
TL;DR: The use of fluorescence energy transfer between an intrinsic energy donor and two chemically added acceptors to study intermediates in the folding of the β2 subunit of E. coli tryptophan‐synthase brings to five the number of intermediates which have been identified and ordered on the folding pathway of the dimeric β2subunit.
Abstract: This report describes the use of fluorescence energy transfer between an intrinsic energy donor (tryptophan 177) and two chemically added acceptors to study intermediates in the folding of the beta 2 subunit of E. coli tryptophan-synthase. Two early folding steps are thus identified and characterized. One is very rapid (its rate constant at 12 degrees C is 0.02 sec-1) and corresponds to the folding of the N-terminal domain into a structure whose overall features approximate well those of the native domain. The second step is somewhat slower (its rate constant at 12 degrees C is 0.008 sec-1) and involves a conformational rearrangement of the N-terminal domain brought about by the interactions between the N- and C-terminal domains within a monomeric beta chain. This brings to five the number of intermediates which have been identified and ordered on the folding pathway of the dimeric beta 2 subunit.

Journal ArticleDOI
01 Feb 1986-Proteins
TL;DR: Trypsin and α‐chymotrypsin were immobilized to alumina‐phosphocolamine complex, activated by glutaraldehyde, and catalyzed transesterification reactions as well as peptide synthesis.
Abstract: Trypsin and alpha-chymotrypsin were immobilized to alumina-phosphocolamine complex, activated by glutaraldehyde. The immobilized enzymes show a great stability toward organic solvents miscible or immiscible with water. In the presence of a low concentration of water, the immobilized enzymes catalyzed transesterification reactions as well as peptide synthesis. The synthesized peptides were stable toward the immobilized enzymes.

Journal ArticleDOI
01 Jan 1986-Proteins
TL;DR: Four major protein sequence data collections have been merged into a single nonredundant data bank called PseqIP, representing most of the available sequence information to date.
Abstract: Four major protein sequence data collections (NBRF-PIR, PSD-Kyoto, PGtrans, and NEWAT) have been merged into a single nonredundant data bank called PseqIP. The data bank entries were automatically matched by a heuristic computer program relying on the fast computation of the number of tetrapeptides shared by two sequences. PseqIP 1.0 includes 6,068 different protein sequences for a total of 1,357,067 residues, representing most of the available sequence information to date. During the course of this work, we found about 600 occurrences course of a protein sequence recorded with a one-amino-acid variation in at least two different data banks. A flat file (ASCII computer-readable format) version of PseqIP 1.0, well-suited for exhaustive homology searches and statistical sequence analysis, is available from our laboratory.