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Biomedical subjects

D S Wishart

Publications and source records attributed to D S Wishart.

15 recordsLinked to original sources
PubMed1996

Modulation of structure and antibacterial and hemolytic activity by ring size in cyclic gramicidin S analogs.

We have evaluated the effect of ring size of gramicidin S analogs on secondary structure, lipid binding, lipid disruption, antibacterial and hemolytic activity. Cyclic analogs with ring sizes ranging from 4 to 14 residues were designed to maintain the amphipathic character as found in gramicidin S and synthesized by solid phase peptide synthesis. The secondary structure of these peptides showed a definite periodicity in beta-sheet content, with rings containing 6, 10, and 14 residues exhibiting beta-sheet structure, and rings containing 8 or 12 residues being largely disordered. Peptides containing 4 or 6 residues did not bind lipopolysaccharide, whereas longer peptides showed a trend of increasing binding affinity for lipopolysaccharide with increasing length. Destabilization of Escherichia coli outer membranes was only observed in peptides containing 10 or more residues. Peptides containing fewer than 10 residues were completely inactive and exhibited no hemolytic activity. The 10-residue peptide showed an activity profile similar to that of gramicidin S itself, with activity against Gram-positive and Gram-negative microorganisms as well as yeast, but also showed high hemolytic activity. Differential activities were obtained by increasing the size of the ring to either 12 or 14 residues. The 14-residue peptide showed no antibiotic activity but exhibited increased hemolytic activity. The 12-residue peptide lost activity against Gram-positive bacteria, retained activity against Gram-negative microorganisms and yeast, but displayed decreased hemolytic activity. Biological activities in the 12-residue peptide were optimized by a series of substitutions in residues comprising both hydrophobic and basic sites resulting in a peptide that exhibited activities comparable with gramicidin S against Gram-negative microorganisms and yeast but with substantially lower hemolytic activity. Compared with gramicidin S, the best analog showed a 10-fold improvement in antibiotic specificity for Gram-negative microorganisms and a 7-fold improvement in specificity for yeast over human erythrocytes as determined by a therapeutic index. These results indicate that it is possible to modulate structure and activities of cyclic gramicidin S analogs by varying ring sizes and further show the potential for developing clinically useful antibiotics based on gramicidin S.

Amino Acid Sequence↗
PubMed1996

Peptide rescue of an N-terminal truncation of the Stoffel fragment of taq DNA polymerase.

Deletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5' to 3' exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N-terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the Stof delta 12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the Stof delta 47, in which 47 amino acids were deleted), resulted in a significant loss of both activity and thermostability. A 33-amino acid synthetic peptide, based on this critical region (i.e., residues 303-335 inclusive), was able to restore 85% of the Stof delta 12 activity when added back to the truncated Stof delta 47 protein as well as return the temperature optimum to that of the Stof delta 12 and Stoffel proteins. Examination of the crystal structure of Taq polymerase (Kim et al., 1995) shows that residues 302-336 of the enzyme form a three-stranded beta-sheet structure that interacts with the remainder of the protein. CD analysis of the 33-amino acid peptide indicates that the free peptide also adopts an ordered structure in solution with more than 50% beta-sheet content. These data suggest that this 33-amino acid peptide constitutes a stable beta-sheet structure capable of rescuing the truncated polymerase in a fashion analogous to the well-documented complementation of Ribonuclease S protein by the 15-residue, alpha-helical, S peptide.

Amino Acid Sequence↗
PubMed1996

Gramicidin S is active against both gram-positive and gram-negative bacteria.

Four linear and four cyclic analogs of gramicidin S (GS) in which D-Phe was replaced with either D-His, D-Ser, D-Tyr or D-Asn have been prepared by solid-phase peptide synthesis and characterized with respect to antibacterial, antifungal and hemolytic activity. Unlike previous reports, GS and a number of cyclic analogs were found to be active against gram-positive as well as gram-negative bacteria. GS showed MICs ranging from 3 to 12.5 micrograms/mL for gram-negative bacteria, compared to MICs of 3 micrograms/mL for gram-positive bacteria. Furthermore, these analogs were also found to exhibit antifungal activity. Unlike the cyclic analogs, all linear analogs were found to be inactive against a wide range of microorganisms tested, and showed low levels of hemolytic activity. The antibacterial activity was found to be highly dependent on the type of assay used, with solution-based assays showing greater activity against gram-negative bacteria than agar-based assays. The GS cyclic analogs were all less toxic than GS itself, with the analog containing the D-Phe to D-Tyr substitution showing the greatest activity of the synthetic analogs. Hemolytic activity in solution against human and sheep red blood cells paralleled antibiotic activity, with those peptides exhibiting greater antibiotic activity generally showing greater hemolytic activity. Membrane destabilization as monitored using the hydrophobic probe N-phenyl-1-naphthylamine was also found to parallel antibacterial and hemolytic activity of cyclic and linear analogs. These results indicate that GS and certain related analogs may have applications as broad-spectrum antibiotics and should be reevaluated for such purposes.

1-Naphthylamine↗
PubMed1996

Epitope mapping of prostate-specific antigen with monoclonal antibodies.

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.

Amino Acid Sequence↗
PubMed1995

1H, 13C and 15N chemical shift referencing in biomolecular NMR.

A considerable degree of variability exists in the way that 1H, 13C and 15N chemical shifts are reported and referenced for biomolecules. In this article we explore some of the reasons for this situation and propose guidelines for future chemical shift referencing and for conversion from many common 1H, 13C and 15N chemical shift standards, now used in biomolecular NMR, to those proposed here.

Carbon Isotopes↗
PubMed1995

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects.

In this study we report on the 1H, 13C and 15N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of 1H, 13C and 15N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strongly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.

Amino Acid Sequence↗
PubMed1994

The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data.

A simple technique for identifying protein secondary structures through the analysis of backbone 13C chemical shifts is described. It is based on the Chemical-Shift Index [Wishart et al. (1992) Biochemistry, 31, 1647-1651] which was originally developed for the analysis of 1H(alpha) chemical shifts. By extending the Chemical-Shift Index to include 13C(alpha), 13C(beta) and carbonyl 13C chemical shifts, it is now possible to use four independent chemical-shift measurements to identify and locate protein secondary structures. It is shown that by combining both 1H and 13C chemical-shift indices to produce a 'consensus' estimate of secondary structure, it is possible to achieve a predictive accuracy in excess of 92%. This suggests that the secondary structure of peptides and proteins can be accurately obtained from 1H and 13C chemical shifts, without recourse to NOE measurements.

Amino Acids↗
PubMed1994

SEQSEE: a comprehensive program suite for protein sequence analysis.

SEQSEE (SEQuence SEEker) is a multi-purpose, menu-driven suite of programs designed to provide a fully integrated, state-of-the-art package for the analysis and display of protein sequences and protein databases. It is currently configured to run on most UNIX-based machines including Sun, SGI and NeXT workstations with conversion to other architectures (e.g. Vax or Cray) being a relatively simple task. SEQSEE is capable of performing nearly all of the analytical and comparative tasks found in most comprehensive commercially available software packages. These include sequence/database searching, sequence retrieval, sequence entry and editing, statistical sequence analysis, multiple sequence alignment, flexible pattern matching, and secondary structure prediction. SEQSEE also integrates a number of unique databases which allow it to perform many additional functions such as structure-based sequence alignments and homology-based secondary structure prediction. Additional enhancements to many previously published algorithms have substantially improved the performance of SEQSEE over that found for most other commercial products. The source code, the documentation and all of the required databases for SEQSEE are freely available and may be obtained by anonymous ftp.

Algorithms↗
PubMed1993

Improved synthetic methods for the selective deuteration of aromatic amino acids: applications of selective protonation towards the identification of protein folding intermediates through nuclear magnetic resonance.

In this report we describe several novel methods for the preparation of selectively deuterated aromatic amino acids. New syntheses for [2,3,5,6-2H4]phenylalanine and [2,4,6,7-2H4]tryptophan, as well as improved catalytic exchange methods for [2,3,5,6-2H4]tyrosine and [2,3,4,5,6-2H5]phenylalanine are presented. Isotopic substitution levels for all compounds are generally found to be greater than 95%. Biosynthetic incorporation of these amino acids is also shown to be possible with little or no evidence of isotopic scrambling. The products from these new syntheses, in combination with other selectively deuterated aromatic amino acids, are found to permit group-specific 'single-proton' labelling of proteins. This highly-efficient and very cost-effective method of selective protonation is shown to produce greatly simplified 1H-NMR spectra of the aromatic region of proteins. The utility of this approach to isotopic editing is demonstrated with the identification of a transient folding intermediate of Escherichia coli thioredoxin which is undetectable by standard 2-D NMR techniques.

Deuterium↗
PubMed1992

The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy.

Previous studies by Wishart et al. [Wishart, D. S., Sykes, B. D., & Richards, F. M. (1991) J. Mol. Biol. (in press)] have demonstrated that 1H NMR chemical shifts are strongly dependent on the character and nature of protein secondary structure. In particular, it has been found that the 1H NMR chemical shift of the alpha-CH proton of all 20 naturally occurring amino acids experiences an upfield shift (with respect to the random coil value) when in a helical configuration and a comparable downfield shift when in a beta-strand extended configuration. On the basis of these observations, a technique is described for rapidly and quantitatively determining the identity, extent, and location of secondary structural elements in proteins based on the simple inspection of the alpha-CH 1H resonance assignments. A number of examples are provided to demonstrate both the simplicity and the accuracy of the technique. This new method is found to be almost as accurate as the more traditional NOE-based methods of determining secondary structure and could prove to be particularly useful in light of the recent development of sequential assignment techniques which are now almost NOE-independent [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667]. We suggest that this new procedure should not necessarily be seen as a substitute to existing rigorous methods for secondary structure determination but, rather, should be viewed as a complement to these approaches.

Amino Acids↗
PubMed1991

Relationship between nuclear magnetic resonance chemical shift and protein secondary structure.

An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships. Similar studies, performed on smaller databases, for 13C and 15N chemical shifts reveal equally strong correlations to protein secondary structure. Among the more interesting results to emerge from this work is the finding that all 20 naturally occurring amino acids experience a mean alpha-1H upfield shift of 0.39 parts per million (from the random coil value) when placed in a helical configuration. In a like manner, the alpha-1H chemical shift is found to move downfield by an average of 0.37 parts per million when the residue is placed in a beta-strand or extended configuration. Similar changes are also found for amide 1H, carbonyl 13C, alpha-13C and amide 15N chemical shifts. Other relationships between chemical shift and protein conformation are also uncovered; in particular, a correlation between helix dipole effects and amide proton chemical shifts as well as a relationship between alpha-proton chemical shifts and main-chain flexibility. Additionally, useful relationships between alpha-proton chemical shifts and backbone dihedral angles as well as correlations between amide proton chemical shifts and hydrogen bond effects are demonstrated.

Hydrogen Bonding↗
PubMed1991

Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy.

Previous work by Wishart et al. (in press) and others [(1989) J. Magn. Reson. 83, 441-449; (1990) J. Magn. Reson. 90, 165-176] has shown a strong tendency for protein secondary structure to be manifested in 1H NMR chemical shifts. Based on these earlier results, two techniques have been developed for the quantification of secondary structure in proteins. Both methods allow for the rapid and accurate determination of the percent content of helix, coil, and beta-strand based on the integration (or peak enumeration) of selected portions of either 1-D or 2-D 1H NMR spectra. These new and very simple procedures have been found to compare quite favorably to other well established techniques for secondary structure determination such as CD, Raman and IR spectroscopy.

Animals↗
PubMed1990

Myxoma virus and malignant rabbit fibroma virus encode a serpin-like protein important for virus virulence.

The leporipoxviruses Shope fibroma virus (SFV), the myxoma virus (MYX), and the SFV/MYX recombinant malignant rabbit fibroma virus (MRV) are closely related yet induce profoundly different diseases in the European rabbit. SFV, which produces a benign tumor at the site of inoculation, is cleared by the immune system after approximately 2 weeks whereas MYX and MRV induce a rapidly lethal systemic infection characterized by generalized suppression of host immune functions. DNA sequencing studies reveal that MRV and MYX possess homologous gene members of the T6/T8/T9 family originally described in the terminal inverted repeat (TIR) of SFV. We also describe a gene present in both MYX and MRV genomes, but which has apparently evolved in the SFV genome into a fragmented pseudogene that appears to contribute to the aggressive nature of MYX and MRV infections. Translation of this open reading frame, designated MYXOMA SERPIN 1 (SERP1), reveals a protein sequence with highly significant homology to the super-family of serine protease inhibitors (serpins) which also includes a number of other poxviral proteins. In the MYX genome the SERP1 gene lies entirely within the TIR sequences and is thus present as two copies, while in the MRV genome SERP1 is present in the unique sequences adjacent to the TIR boundary and hence is a single copy. The amino acid homology between the putative active site of SERP1 and those of other serpins predicts that the target enzyme will be different from the known catalog of serine antiprotease substrates. Deletion of this gene from MRV significantly attenuates the disease spectrum induced by the normally lethal virus. Although the MRV-S1 deletion construct (MRV with SERP1 gene deleted) grows in all tissue culture cells tested in a fashion identical to the MRV parent, the majority of rabbits infected with MRV-S1 are able to mount an effective immune response and totally recover from the virus infection to become resistant to subsequent challenge by MRV or MYX.

Amino Acid Sequence↗