Search PubMed⌕ Search

Biomedical subjects

S A Benner

Publications and source records attributed to S A Benner.

At least 73 records · Page 4Linked to original sources
PubMed1994

Predicting the conformation of proteins from sequences. Progress and future progress.

A new paradigm for predicting the secondary and tertiary structure of functional proteins from sequence data has emerged from detailed models of how natural selection, conservation, and neutral drift, the three fundamental factors in molecular evolution, leave their mark upon protein sequences. Structural information is extracted from a set of aligned homologous sequences via an analysis of patterns of conservation and variation between proteins with quantitatively defined evolutionary relationships. Tertiary structural information is obtained prior to the assignment of secondary structure, where it plays an important role. Throughout, structural predictions are made with the active involvement of a biochemist whose expertise and insight is critical both for making the prediction and in analyzing its successful and unsuccessful parts. Secondary structure predictions are evaluated based on their ability to sustain an effort to model tertiary structure. Several predictions made using the new paradigm can now be compared with those made under the classical paradigm, including a neural network. The results obtained from the new paradigm are clearly superior to those obtained with the classical paradigm, at least within the protein families that were examined.

Amino Acid Sequence↗
PubMed1994

Expanding the genetic lexicon: incorporating non-standard amino acids into proteins by ribosome-based synthesis.

Only 20 amino acids are normally incorporated into proteins synthesized in living cells, and this has limited the structural range of proteins that can be prepared. New methods that allow the incorporation of amino acids that are not normally encoded by natural genes are being developed: these include reassigning functions within the existing genetic code, and expanding the genetic code by constructing additional, non-natural codons. Used in conjunction with recent major advances in understanding protein structure-function relationships, these approaches should extend the range of de novo protein designs that are possible.

Amino Acid Sequence↗
PubMed1994

Amino acid substitution during functionally constrained divergent evolution of protein sequences.

In aligning homologous protein sequences, it is generally assumed that amino acid substitutions subsequent in time occur independently of amino acid substitutions previous in time, i.e. that patterns of mutation are similar at low and high sequence divergence. This assumption is examined here and shown to be incorrect in an interesting way. Separate mutation matrices were constructed for aligned protein sequence pairs at divergences ranging from 5 to 100 PAM units (point accepted mutations per 100 aligned positions). From these, the corresponding log-odds (Dayhoff) matrices, normalized to 250 PAM units, were constructed. The matrices show that the genetic code influences accepted point mutations strongly at early stages of divergence, while the chemical properties of the side chains dominate at more advanced stages.

Amino Acid Sequence↗
PubMed1993

Synthesis, structure and activity of artificial, rationally designed catalytic polypeptides.

Biological macromolecules with catalytic activity can be created artificially using two approaches. The first exploits a system that selects a few catalytically active biomolecules from a large pool of randomly generated (and largely inactive) molecules. Catalytic antibodies and many catalytic RNA molecules are obtained in this way. The second involves rational design of a biomolecule that folds in solution to present to the substrate an array of catalytic functional groups. Here we report the synthesis of rationally designed polypeptides that catalyse the decarboxylation of oxaloacetate via an imine intermediate. We determine the secondary structures of the polypeptides by two-dimensional NMR spectroscopy. We are able to trap and identify intermediates in the catalytic cycle, and to explore the kinetics in detail. The formation of the imine by our artificial oxaloacetate decarboxylases is three to four orders of magnitude faster than can be achieved with simple amine catalysts: this performance rivals that of typical catalytic antibodies.

Amines↗
PubMed1993

Enzymatic recognition of the base pair between isocytidine and isoguanosine.

The ability of various polymerases to catalyze the template-directed formation of a base pair between isoguanine (iso-G) and isocytosine (iso-C) in duplex oligonucleotides has been investigated. A new procedure was developed for preparing derivatives of deoxyisoguanosine suitable for incorporation into DNA using an automated DNA synthesizer. T7 RNA polymerase, AMV reverse transcriptase, and the Klenow fragment of DNA polymerase all incorporated iso-G opposite iso-C in a template. T4 DNA polymerase did not. Several polymerases also incorporated iso-G opposite T, presumably through pairing with a minor tautomeric form of iso-G complementary to T. In a template, iso-G directs the incorporation of both iso-C and T when Klenow fragment is the catalyst and only U when T7 RNA polymerase is the catalyst. Further, derivatives of iso-C were found to undergo significant amounts of deamination under alkaline conditions used for base deprotection after automated oligonucleotide synthesis. Both the deamination reaction of iso-C and the ambivalent tautomeric forms of iso-G make it unlikely that the (iso-C).(iso-G) base pair was a part of information storage molecules also containing the A.T and G.C base pairs found in primitive forms of life that emerged on planet earth several billion years ago. Nevertheless, the extra letters in the genetic alphabet can serve useful roles in a contemporary laboratory setting.

Adenosine↗
PubMed1993

A secondary structure prediction of the hemorrhagic metalloprotease family.

A secondary structure has been predicted for the hemorrhagic metalloproteases using a method developed in Zurich that extracts structural information from patterns of conservation and variation in homologous protein sequences. This prediction tests the limits of the method when applied to a small number of homologous sequences that have undergone only modest evolutionary divergence. Predictions were also obtained using a neural network developed by Sander and coworkers, to date the best fully automated method for predicting secondary structure, and using the classical Chou-Fasman and GOR heuristics. The predictions are different. No crystal structure is known within this protein family, but one is expected shortly. Therefore, this prediction should contribute significantly to the evaluation of the relative merits of these prediction methods.

Amino Acid Sequence↗
PubMed1993

Predicting the conformation of proteins. Man versus machine.

Two types of approaches for predicting the conformation of proteins from sequence data have lately received attention: 'black box' tools that generate fully automated predictions of secondary structure from a set of homologous protein sequences, and methods involving the expertise of a human biochemist who is assisted, but not replaced, by computer tools. A friendly controversy has emerged as to which approach offers a brighter future. In fact, both are necessary. Nevertheless, a snapshot of the controversy at this instant offers much insight into the structure prediction problem itself.

Amino Acid Sequence↗
PubMed1993

The nitrogenase MoFe protein. A secondary structure prediction.

Surface residues, interior residues, and parsing residues, together with a secondary structure derived from these, are predicted for the MoFe nitrogenase protein in advance of a crystal structure of the protein, scheduled shortly to appear in Nature. By publishing this prediction, we test our method for predicting the conformation of proteins from patterns in the divergent evolution of homologous protein sequences in a way that places the method 'at risk'.

Amino Acid Sequence↗
PubMed1993

Empirical and structural models for insertions and deletions in the divergent evolution of proteins.

The exhaustive matching of the protein sequence database makes possible a broadly based study of insertions and deletions (indels) during divergent evolution. In this study, the probability of a gap in an alignment of a pair of homologous protein sequences was found to increase with the evolutionary distance measured in PAM units (number of accepted point mutations per 100 amino acid residues). A relationship between the average number of amino acid residues between indels and evolutionary distance suggests that a unit 30 to 40 amino acid residues in length remains, on average, undisrupted by indels during divergent evolution. Further, the probability of a gap was found to be inversely proportional to gap length raised to the 1.7 power. This empirical law fits closely over the entire range of gap lengths examined. Gap length distribution is largely independent of evolutionary distance. These results rule out the widely used linear gap penalty as a satisfactory formula for scoring gaps when constructing alignments. Further, the observed gap length distribution can be explained by a simple model of selective pressures governing the acceptance of indels during divergent evolution. Finally, this model provides theoretical support for using indels as part of "parsing algorithms", important in the de novo prediction of the folded structure of proteins from the sequence data.

Amino Acid Sequence↗
PubMed1993

Predicted secondary structure for the Src homology 3 domain.

A de novo secondary structure prediction has been prepared for Src homology domain 3, in advance of any crystallographic information concerning any member of this interesting protein family. The prediction can be compared with a crystal structure that will be published in Nature on October 29, 1992. The prediction is based on analysis of a multiple alignment of homologous proteins. The patterns of variation and conservation of amino acids across the alignment allow the determination of surface and internal positions, which then allow the assignment of secondary structure. The prediction is quite different both in method and, in this case, result from predictions based on propensities (e.g. Garnier-Osgurthorpe-Robson) of particular amino acids to appear in particular types of secondary structure.

Amino Acid Sequence↗
PubMed1993

Oligonucleotide analogs with dimethylenesulfide, -sulfoxide, and -sulfone groups replacing phosphodiester linkages.

Two sets of experimental protocols are given for the synthesis of 3',5'-bis-homodeoxyribonucleosides, building blocks for the synthesis of oligodeoxynucleotide analogs where the -O-PO2-O- groups are replaced by -CH2-S-CH2-, -CH2-SO-CH2-, and -CH2-SO2-CH2- units. Conditions are presented for joining these building blocks to create short nucleic acid analogs. Since isosteric, achiral, and non-ionic analogs of natural oligonucleotides stable to both enzymatic and chemical hydrolysis, such molecules have potential application as probes in the laboratory, in studies of the biological function of individual genes, and as "antisense" oligonucleotide analogs.

Acrylates↗
PubMed1992

Exhaustive matching of the entire protein sequence database.

The entire protein sequence database has been exhaustively matched. Definitive mutation matrices and models for scoring gaps were obtained from the matching and used to organize the sequence database as sets of evolutionarily connected components. The methods developed are general and can be used to manage sequence data generated by major genome sequencing projects. The alignments made possible by the exhaustive matching are the starting point for successful de novo prediction of the folded structures of proteins, for reconstructing sequences of ancient proteins and metabolisms in ancient organisms, and for obtaining new perspectives in structural biochemistry.

Amino Acid Sequence↗
PubMed1992

Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code.

One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role.

Amino Acid Sequence↗
PubMed1991

A C-nucleotide base pair: methylpseudouridine-directed incorporation of formycin triphosphate into RNA catalyzed by T7 RNA polymerase.

With templates containing 2'-deoxy-1-methylpseudouridine (dm psi), T7 RNA polymerase catalyzes the incorporation of either adenosine triphosphate (ATP) or formycin triphosphate (FTP) into a growing chain of RNA with the same efficiency as with templates containing thymidine (dT). In each case, the overall rate of synthesis of full-length products containing formycin is about one-tenth of the rate of synthesis of analogous products containing adenosine. Analysis of the products of abortive initiation shows that incorporation of FMP into the growing oligonucleotide by T7 RNA polymerase is more likely to lead to premature termination of transcription than is incorporation of AMP. Nevertheless, the results demonstrate that T7 RNA polymerase tolerates the formation of a C-nucleotide transcription complex in which the nucleoside bases on both the template and the incoming nucleotide are joined to the ribose by a carbon-carbon bond. This result increases the prospects for further expanding the genetic alphabet via incorporation of new base pairs with novel hydrogen-bonding schemes (Piccirilli et al., 1990).

Adenosine Triphosphate↗
PubMed1991

Structural determinants of stereospecificity in yeast alcohol dehydrogenase.

Replacing Leu-182 by Ala in yeast alcohol dehydrogenase (YADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) yields a mutant that retains 34% of its kcat value and makes one stereochemical "mistake" every 850,000 turnovers (instead of approximately 1 error every 7,000,000,000 turnovers in native YADH) in its selection of the 4-Re hydrogen of NADH. Half of the decrease in stereochemical fidelity comes from an increase in the rate of transfer of the 4-Si hydrogen of NADH. The mutant also accepts 5-methylnicotinamide adenine dinucleotide, a cofactor analog not accepted by native YADH. The stereospecificity of the mutant is lower still with analogs of NADH where the carboxamide group of the nicotinamide ring is replaced by groups with weaker hydrogen bonding potential. For example, with thio-NADH, the mutant enzyme makes 1 stereochemical "mistake" every 450 turnovers. Finally, the double mutant T157S/L182A, in which Thr-157 is replaced by Ser and Leu-182 is replaced by Ala, also shows decreased stereochemical fidelity. These results suggest that Si transfer in the mutant enzymes arises from NADH bound in a syn conformation in the active site and that this binding is not obstructed in native YADH by side chains essential for catalysis.

Alcohol Dehydrogenase↗