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S B Kent

Publications and source records attributed to S B Kent.

At least 55 records · Page 3Linked to original sources
PubMed1995

Analysis of the proteolytic processing and activation of the rice tungro bacilliform virus reverse transcriptase.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and member of the badnavirus subgroup. Open reading frame (ORF) 3 encodes the viral capsid protein, protease (PR), and reverse transcriptase (RT). A DNA fragment of ORF 3 that contains PR and RT sequences was previously expressed in insect cells to produce the PR/RT polyprotein that was processed to yield p62 and p55. p62 and p55 share common N-terminal amino acid sequences and exhibit reverse transcriptase activity. Mass spectrometry was employed to determine the precise molecular weight of the p62 and p55 proteins and enabled determination of the C-termini for both proteins. ORFs encoding either p62 or p55 were constructed and expressed in insect cells using the baculoviruses 62R-BBac and 55R-BBac, respectively. The recombinant p62R and p55R proteins were purified separately and shown to have the same enzymatic activities as previously reported for the processed p62 and p55. The putative active site of the PR was mutated (mpr), and the resulting mpr/RT ORF was expressed in insect cells using the baculovirus mpr/RT-BBac. The mpr/RT polyprotein was not processed in insect cells, resulting in the accumulation of the approximately 87-kDa mpr/RT polyprotein. This study further extends the understanding of p62 and p55 and clarifies the role of the RTBV PR in processing of the RT.

Animals↗
PubMed1995

Detection of synthetic protein isomers and conformers by electrospray mass spectrometry.

Electrospray mass spectrometry (ESMS) has been used to investigate the structural properties of a protein prepared by total chemical synthesis. Construction of an analog of the tenth type III module from fibronectin (10F3) by chemical ligation of the unprotected synthetic peptides 10F3(1-40) alpha COSH and BrAc (42-94) 10F3 was found to give two major products, both of which possessed a mass corresponding to the expected product, [(COS)40-41]10F3. Comparisons of the ESMS charge distributions obtained for these two synthetic products with that obtained for recombinant 10F3 suggested that one of the synthetic 10F3 analogs was correctly folded and the other was somehow misfolded. This was further confirmed by 1D and 2D NMR analysis. Exposure of the misfolded synthetic [(COS)40-41]10F3 to high pH and elevated temperature followed by analysis using liquid chromatography-mass spectrometry revealed a beta-ester linkage between residues Asn42 and Ser43, produced by an N-->O acyl shift rearrangement at Ser43, as the origin of the misfolding. ESMS was also used to measure the H-D exchange rates of labile protons within the synthetic and recombinant 10F3s. This application, which allows the number of slow exchanging backbone amides within a protein to be calculated, revealed clear differences in the H-bonding networks of the folded and unfolded synthetic protein modules. Replacement of Ser43 by an alanine was found to circumvent the N-->O acyl shift, and the resulting synthetic protein analogue, [Ala43, (COS)40-41]10F3, possessed identical structural properties to recombinant 10F3.

Amino Acid Sequence↗
PubMed1994

Synthesis of proteins by native chemical ligation.

A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chemical ligation was demonstrated by the one-step preparation of a cytokine containing multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-containing protein molecule. Native chemical ligation is an important step toward the general application of chemistry to proteins.

Amino Acid Sequence↗
PubMed1994

Design and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: integrin alpha IIb beta 3 (platelet GPIIb-IIIa).

Integrins are a class of heterodimeric cell adhesion receptors involved in cell migration, cell anchorage, and cell-cell interactions. The cytoplasmic domains of integrins are of key importance in these activities. We have designed and chemically synthesized a 126 amino acid model protein (MP-1) containing both cytoplasmic tails of the platelet-derived integrin alpha IIb beta 3 covalently linked via a helical coiled coil. The coiled-coil tertiary structure was incorporated to mimic the membrane-spanning domain of the integrin and to act as a topological constraint fixing the two cytoplasmic tails in a parallel arrangement. This molecule, which contains two C-termini, was constructed by chemical dovetailing. The bromoacetylated and cysteinyl peptide synthons were unambiguously ligated through the formation of a thioether linkage. Ultraviolet circular dichroism (CD) spectroscopy has been performed on MP-1 and related compounds, confirming that a helical coiled coil is present within the MP-1 molecule. Significantly, the helicity apparently extends beyond the predicted amphiphilic region of MP-1. Fluorescence measurements suggest that a defined tertiary structure has formed by the association of the two cytoplasmic domains. We conclude that this is a practical design strategy for the study of the cytoplasmic domain of multisubunit cell-surface receptors.

Amino Acid Sequence↗
PubMed1994

The primary structure of halocyanin, an archaeal blue copper protein, predicts a lipid anchor for membrane fixation.

Halocyanin, a small blue copper protein, was isolated from the haloalkaliphilic archaeon Natronobacterium pharaonis. The NH2 terminus was not accessible to Edman degradation. About 70% of the amino acid sequence was determined by protein sequence analysis. The sequence information of two peptides was used for cloning and sequencing the halocyanin gene (hcy). The open reading frame codes for 489 base pairs, which account for a protein with 163 amino acids and a molecular mass of 17,223 Da. The discrepancy between this value and the molecular mass of 15,456 +/- 1.5 Da for the copper-free protein determined by electrospray mass spectrometry can be explained by a post-translational processing of the gene product. The NH2-terminal sequence of the open reading frame contains a motif that is characteristic for prokaryotic lipoproteins. Assuming a similar processing for halocyanin, Cys at position 25 of the primary transcript would be modified by a diphytanyl (glycerol)diether. Subsequently, the precursor is cleaved by a signal peptidase II-like protease and then acetylated at its NH2-terminal alpha-amino group. These modifications would yield a protein with a calculated molecular mass of 15,456 Da. A comparison of the primary structure of halocyanin with a number of other blue copper proteins places it into the plastocyanin-related group.

Amino Acid Sequence↗
PubMed1994

An 1H NMR determination of the three-dimensional structures of mirror-image forms of a Leu-5 variant of the trypsin inhibitor from Ecballium elaterium (EETI-II).

The 3-dimensional structures of mirror-image forms of a Leu-5 variant of the trypsin inhibitor Ecballium elaterium (EETI-II) have been determined by 1H NMR spectroscopy and simulated annealing calculations incorporating NOE-derived distance constraints. Spectra were assigned using 2-dimensional NMR methods at 400 MHz, and internuclear distances were determined from NOESY experiments. Three-bond spin-spin couplings between C alpha H and amide protons, amide exchange rates, and the temperature dependence of amide chemical shifts were also measured. The structure consists largely of loops and turns, with a short region of beta-sheet. The Leu-5 substitution produces a substantial reduction in affinity for trypsin relative to native EETI-II, which contains an Ile at this position. The global structure of the Leu-5 analogue studied here is similar to that reported for native EETI-II (Heitz A, Chiche L, Le-Nguyen D, Castro B, 1989, Biochemistry 28:2392-2398) and to X-ray and NMR structures of the related proteinase inhibitor CMTI-I (Bode W et al., 1989, FEBS Lett 242:285-292; Holak TA et al., 1989a, J Mol Biol 210:649-654; Holak TA, Gondol D, Otlewski J, Wilusz T, 1989b, J Mol Biol 210:635-648; Holak TA, Habazettl J, Oschkinat H, Otlewski J, 1991, J Am Chem Soc 113:3196-3198). The region near the scissile bond is the most disordered part of the structure, based on geometric superimposition of 40 calculated structures. This disorder most likely reflects additional motion being present in this region relative to the rest of the protein. This motional disorder is increased in the Leu-5 analogue relative to the native form and may be responsible for its reduced trypsin binding. A second form of the protein synthesized with all (D) amino acids was also studied by NMR and found to have a spectrum identical with that of the (L) form. This is consistent with the (D) form being a mirror image of the (L) form and not distinguishable by NMR in an achiral solvent (i.e., H2O). The (D) form has no activity against trypsin, as would be expected for a mirror-image form.

Amino Acid Sequence↗
PubMed1993

Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis.

An analogue of "HIV-1 protease" was designed in which the ability to donate important water-mediated hydrogen bonds to substrate was precisely and directly deleted. Chemical ligation of unprotected peptide segments was used to synthesize this "backbone-engineered" enzyme. The functionally relevant amide -CONH- linkage between residues Gly49-Ile50 in each flap of the enzyme was replaced by an isosteric thioester -COS- bond. The backbone-engineered enzyme had normal substrate specificity and affinity (Km). However, the catalytic activity (kcat) was reduced approximately 3000-fold compared to the native amide bond-containing enzyme. Inhibition by the reduced peptide bond substrate analogue MVT-101 was unaffected compared with native enzyme. By contrast, the normally tight-binding hydroxyethylamine inhibitor JG-365 bound to the backbone-engineered enzyme with an approximately 2500-fold reduction in affinity. The reduced catalytic activity of the -Gly49-psi(COS)-Ile50-backbone-engineered enzyme analogue provides direct experimental evidence to support the suggestion that backbone hydrogen bonds from the enzyme flaps to the substrate are important for the catalytic function of the HIV-1 protease.

Amino Acid Sequence↗
PubMed1993

Protein ladder sequencing.

A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost.

Amino Acid Sequence↗
PubMed1993

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.

Amino Acid Sequence↗
PubMed1993

The chemical synthesis of proteins.

Total chemical synthesis is developing as an important approach to the construction of native proteins and their analogs for the study of the structural basis of biochemical activity. In the past year, a radical departure from conventional synthetic approaches has led to a renaissance in this field. The inherent simplicity of these new modes of protein construction will allow large synthetic proteins (> 200 amino acids in length) to be routinely assembled; this will vastly expand the repertoire of protein molecules accessible to research.

Biotechnology↗
PubMed1993

Identification of proteolytic processing sites within the Gag and Pol polyproteins of feline immunodeficiency virus.

N-terminal amino acid sequencing, ion spray mass spectrometry, and cleavage of synthetic peptide substrates were used to identify the N and C termini of the mature Gag and Pol proteins of feline immunodeficiency virus (FIV). The Gag polyprotein encodes matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. The Gag-Pol polyprotein encodes, in addition to the above proteins, protease (PR), reverse transcriptase (RT), dUTPase (DU), and integrase (IN). Secondary cleavage of RT at Trp-595-Tyr-596 of Pol yields a truncated form lacking the C-terminal RNase H domain. The observed and expected molecular masses of the viral proteins were in agreement, with three exceptions. (i) The molecular mass of MA was 14,735 Da, compared with a predicted mass of 14,649 Da, based on a single cleavage at Tyr-135-Pro-136 of Gag. The observed molecular mass is consistent with myristoylation of MA, which was confirmed by metabolic labeling of FIV MA with [3H]myristic acid. (ii) The N terminus of the NC protein is generated via cleavage at Gln-366-Val-367 of Gag, which predicts a mass of 25,523 for CA and 9,101 for the major form of NC. The observed mass of CA was 24,569, consistent with loss of nine C-terminal amino acids by a second cleavage of CA at Leu-357-Leu-358. Synthetic FIV protease accurately cleaved synthetic peptide substrates containing this site. (iii) The actual mass of NC (7,120 Da) was approximately 2 kDa smaller than the mass predicted by synthesis to the stop codon at the end of Gag (9,101 Da). Experiments are in progress to characterize additional cleavage(s) in NC.

Amino Acid Sequence↗
PubMed1992

Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins.

Two new technologies have made the study of proteins by mass spectrometry straight-forward. Proteins with molecular masses of up to more than 100 kilodaltons can be analyzed at picomole sensitivities to give simple mass spectra corresponding to the intact molecule. This development has allowed unprecedented accuracy in the determination of the molecular weights of proteins. A number of "case studies" are used to present the revolutionary impact that these powerful new ways of looking at proteins are having on biological research.

Amino Acid Sequence↗
PubMed1992

Ion-spray tandem mass spectrometry in peptide synthesis: structural characterization of minor by-products in the synthesis of ACP(65-74).

Ion-spray triple quadrupole mass spectrometry was used to investigate the products from the solid phase synthesis of the decapeptide (H)-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-(OH) [acyl carrier protein(65-74)]. The target sequence was assembled in stepwise fashion from the C-terminal using Boc chemistry on a Bly-OCH2-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with HF/p-cresol for 1 h at 0 degrees C. The crude product was analyzed by reverse-phase HPLC and contained a single major peptide component, one significant minor (late-eluting) component and several trace-level peptide by-products. The components were separated by HPLC and the fractions directly analyzed by mass spectrometry and tandem mass spectrometry. The major product was confirmed as the desired ACP(65-74). The significant minor component was apparently from incomplete deprotection of Asp70, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp70, succinimide formation at Asn73, acylation of the Tyr71 side chain phenolic hydroxyl leading to a branched heptadecapeptide, and tert-butylation of the decapeptide. The possible origins of these by-products are discussed in light of known peptide chemistry. Also notable was the absence, to very low detection levels, of by-products frequently reported to occur in peptide synthesis, illustrating the high degree of refinement and the accuracy of currently used synthetic methods.

Acyl Carrier Protein↗
PubMed1992

Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected].

The D and L forms of the enzyme HIV-1 protease have been prepared by total chemical synthesis. The two proteins had identical covalent structures. However, the folded protein-enzyme enantiomers showed reciprocal chiral specificity on peptide substrates. That is, each enzyme enantiomer cut only the corresponding substrate enantiomer. Reciprocal chiral specificity was also evident in the effect of enantiomeric inhibitors. These data imply that the folded forms of the chemically synthesized D- and L-enzyme molecules are mirror images of one another in all elements of the three-dimensional structure. Enantiomeric proteins are expected to display reciprocal chiral specificity in all aspects of their biochemical interactions.

Amino Acid Sequence↗
PubMed1992

Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease.

Backbone-engineered HIV-1 protease was prepared by a total chemical synthesis approach that combines the act of joining two peptides with the generation of an analog structure. Unprotected synthetic peptide segments corresponding to the two halves of the HIV-1 protease monomer polypeptide chain were joined cleanly and in high yield through unique mutually reactive functional groups, one on each segment. Ligation was performed in 6 molar guanidine hydrochloride, thus circumventing limited solubility of protected peptide segments, the principal problem of the classical approach to the chemical synthesis of proteins. The resulting fully active HIV-1 protease analog contained a thioester replacement for the natural peptide bond between Gly51-Gly52 in each of the two active site flaps, a region known to be highly sensitive to mutational changes of amino acid side chains.

Amino Acid Sequence↗
PubMed1991

Inhibition of intercellular adhesion molecule 1-dependent biological activities by a synthetic peptide analog.

We have used a combination of hydropathy analysis of the intercellular adhesion molecule 1 (ICAM-1) sequence and dot-matrix comparison of the sequence with the homologous, but functionally distinct, protein myelin-associated glycoprotein to identify a putative functional binding region. One polar, and presumably surface-exposed, region of ICAM-1 showed no significant identity with myelin-associated glycoprotein. A synthetic peptide analog based on the sequence of this region (JF9) mimicked the inhibitory effects of the anti-ICAM-1 monoclonal antibody WEHI-CAM-1. These included inhibition of ICAM-1-dependent homotypic aggregation of Raji Burkitt lymphoma and phorbol-ester treated U937 cells at concentrations as low as 80 micrograms/ml (24 microM). In addition, at a concentration of 100 micrograms/ml, the peptide analog effectively inhibited cytotoxic cell activity, an ICAM-1-dependent effector function of the immune response. This simple method of sequence analysis may have general applicability to the identification of functional domains in homologous, but functionally distinct, proteins such as the translated products of gene families.

Amino Acid Sequence↗
PubMed1991

Structure at 2.5-A resolution of chemically synthesized human immunodeficiency virus type 1 protease complexed with a hydroxyethylene-based inhibitor.

The crystal structure of a complex between chemically synthesized human immunodeficiency virus type 1 (HIV-1) protease and an octapeptide inhibitor has been refined to an R factor of 0.138 at 2.5-A resolution. The substrate-based inhibitor, H-Val-Ser-Gln-Asn-Leu psi [CH(OH)CH2]Val-Ile-Val-OH (U-85548e) contains a hydroxyethylene isostere replacement at the scissile bond that is believed to mimic the tetrahedral transition state of the proteolytic reaction. This potent inhibitor has Ki less than 1 nM and was developed as an active-site titrant of the HIV-1 protease. The inhibitor binds in an extended conformation and is involved in beta-sheet interactions with the active-site floor and flaps of the enzyme, which form the substrate/inhibitor cavity. The inhibitor diastereomer has the S configuration at the chiral carbon atom of the hydroxyethylene insert, and the hydroxyl group is within H-bonding distance of the two active-site carboxyl groups in the enzyme dimer. The two subunits of the enzyme are related by a pseudodyad, which superposes them at a 178 degrees rotation. The main difference between the subunits is in the beta turns of the flaps, which have different conformations in the two monomers. The inhibitor has a clear preferred orientation in the active site and the alternative conformation, if any, is a minor one (occupancy of less than 30%). A new model of the enzymatic mechanism is proposed in which the proteolytic reaction is viewed as a one-step process during which the nucleophile (water molecule) and electrophile (an acidic proton) attack the scissile bond in a concerted manner.

Amino Acid Sequence↗