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Biomedical subjects

S L Mayo

Publications and source records attributed to S L Mayo.

32 records · Page 2Linked to original sources
PubMed1998

Pairwise calculation of protein solvent-accessible surface areas.

BACKGROUND: The tractability of many algorithms for determining the energy state of a system depends on the pairwise nature of an energy expression. Some energy terms, such as the standard implementation of the van der Waals potential, satisfy this criterion whereas others do not. One class of important potentials that are not pairwise involves benefits and penalties for burying hydrophobic and/or polar surface areas. It has been found previously that, in some cases, a pairwise approximation to these surface areas correlates with the true surface areas. We set out to generalize the applicability of this approximation. RESULTS: We develop a pairwise expression with one scalable parameter that closely reproduces both the true buried and the true exposed solvent-accessible surface areas. We then refit our previously published coiled-coil stability data to give solvation parameters of 26 cal/mol A2 favoring hydrophobic burial and 100 cal/mol A2 opposing polar burial. CONCLUSIONS: An accurate pairwise approximation to calculate exposed and buried protein solvent-accessible surface area is achieved.

Algorithms↗
PubMed1998

Design, structure and stability of a hyperthermophilic protein variant.

Here we report the use of an objective computer algorithm in the design of a hyperstable variant of the Streptococcal protein Gbeta1 domain (Gbeta1). The designed seven-fold mutant, Gbeta1-c3b4, has a melting temperature in excess of 100 degrees C and an enhancement in thermodynamic stability of 4.3 kcal mol(-1) at 50 degrees C over the wild-type protein. Gbeta1-c3b4 maintains the Gbeta1 fold, as determined by nuclear magnetic resonance spectroscopy, and also retains a significant level of binding to human IgG in qualitative comparisons with wild type. The basis of the stability enhancement appears to have multiple components including optimized core packing, increased burial of hydrophobic surface area, more favorable helix dipole interactions, and improvement of secondary structure propensity. The design algorithm is able to model such complex contributions simultaneously using empirical physical/chemical potential functions and a combinatorial optimization algorithm based on the dead-end elimination theorem. Because the design methodology is based on general principles, there is the potential of applying the methodology to the stabilization of other unrelated protein folds.

Algorithms↗
PubMed1997

De novo protein design: towards fully automated sequence selection.

Several groups have applied and experimentally tested systematic, quantitative methods to protein design with the goal of developing general design algorithms. We have sought to expand the range of computational protein design by developing quantitative design methods for residues of all parts of a protein: the buried core, the solvent exposed surface, and the boundary between core and surface. Our goal is an objective, quantitative design algorithm that is based on the physical properties that determine protein structure and stability and which is not limited to specific folds or motifs. We chose the betabetaalpha motif typified by the zinc finger DNA binding module to test our design methodology. Using previously published sequence scoring functions developed with a combined experimental and computational approach and the Dead-End Elimination theorem to search for the optimal sequence, we designed 20 out of 28 positions in the test motif. The resulting sequence has less than 40% homology to any known sequence and does not contain any metal binding sites or cysteine residues. The resulting peptide, pda8d, is highly soluble and monomeric and circular dichroism measurements showed it to be folded with a weakly cooperative thermal unfolding transition. The NMR solution structure of pda8d was solved and shows that it is well-defined with a backbone ensemble rms deviation of 0. 55 A. Pda8d folds into the desired betabetaalpha motif with well-defined elements of secondary structure and tertiary organization. Superposition of the pda8d backbone to the design target is excellent, with an atomic rms deviation of 1.04 A.

Algorithms↗
PubMed1997

De novo protein design: fully automated sequence selection.

The first fully automated design and experimental validation of a novel sequence for an entire protein is described. A computational design algorithm based on physical chemical potential functions and stereochemical constraints was used to screen a combinatorial library of 1.9 x 10(27) possible amino acid sequences for compatibility with the design target, a betabetaalpha protein motif based on the polypeptide backbone structure of a zinc finger domain. A BLAST search shows that the designed sequence, full sequence design 1 (FSD-1), has very low identity to any known protein sequence. The solution structure of FSD-1 was solved by nuclear magnetic resonance spectroscopy and indicates that FSD-1 forms a compact well-ordered structure, which is in excellent agreement with the design target structure. This result demonstrates that computational methods can perform the immense combinatorial search required for protein design, and it suggests that an unbiased and quantitative algorithm can be used in various structural contexts.

Algorithms↗
PubMed1997

Probing the role of packing specificity in protein design.

By using a protein-design algorithm that quantitatively considers side-chain packing, the effect of specific steric constraints on protein design was assessed in the core of the streptococcal protein G beta1 domain. The strength of packing constraints used in the design was varied, resulting in core sequences that reflected differing amounts of packing specificity. The structural flexibility and stability of several of the designed proteins were experimentally determined and showed a trend from well-ordered to highly mobile structures as the degree of packing specificity in the design decreased. This trend both demonstrates that the inclusion of specific packing interactions is necessary for the design of native-like proteins and defines a useful range of packing specificity for the design algorithm. In addition, an analysis of the modeled protein structures suggested that penalizing for exposed hydrophobic surface area can improve design performance.

Algorithms↗
PubMed1997

Automated design of the surface positions of protein helices.

Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.

Algorithms↗
PubMed1997

Coupling backbone flexibility and amino acid sequence selection in protein design.

Using a protein design algorithm that considers side-chain packing quantitatively, the effect of explicit backbone motion on the selection of amino acids in protein design was assessed in the core of the streptococcal protein G beta 1 domain (G beta 1). Concerted backbone motion was introduced by varying G beta 1's supersecondary structure parameter values. The stability and structural flexibility of seven of the redesigned proteins were determined experimentally and showed that core variants containing as many as 6 of 10 possible mutations retain native-like properties. This result demonstrates that backbone flexibility can be combined explicitly with amino acid side-chain selection and that the selection algorithm is sufficiently robust to tolerate perturbations as large as 15% of G beta 1's native supersecondary structure parameter values.

Algorithms↗
PubMed1997

Circular dichroism determination of class I MHC-peptide equilibrium dissociation constants.

Class I major histocompatibility complex (MHC) molecules bind peptides derived from degraded proteins for display to T cells of the immune system. Peptides bind to MHC proteins with varying affinities, depending upon their sequence and length. We demonstrate that the thermal stability of the MHC-peptide complex depends directly on peptide binding affinity. We use this correlation to develop a convenient method to determine peptide dissociation constants by measuring MHC-peptide complex stability using thermal denaturation profiles monitored by circular dichroism.

Amino Acid Sequence↗
PubMed1996

Protein design automation.

We have conceived and implemented a cyclical protein design strategy that couples theory, computation, and experimental testing. The combinatorially large number of possible sequences and the incomplete understanding of the factors that control protein structure are the primary obstacles in protein design. Our protein design automation algorithm objectively predicts protein sequences likely to achieve a desired fold. Using a rotamer description of the side chains, we implemented a fast discrete search algorithm based on the Dead-End Elimination Theorem to rapidly find the globally optimal sequence in its optimal geometry from the vast number of possible solutions. Rotamer sequences were scored for steric complementarity using a van der Waals potential. A Monte Carlo search was then executed, starting at the optimal sequence, in order to find other high-scoring sequences. As a test of the design methodology, high-scoring sequences were found for the buried hydrophobic residues of a homodimeric coiled coil based on GCN4-p1. The corresponding peptides were synthesized and characterized by CD spectroscopy and size-exclusion chromatography. All peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures ranging from 24 degrees C to 57 degrees C. A quantitative structure activity relation analysis was performed on the designed peptides, and a significant correlation was found with surface area burial. Incorporation of a buried surface area potential in the scoring of sequences greatly improved the correlation between predicted and measured stabilities and demonstrated experimental feedback in a complete design cycle.

Algorithms↗
PubMed1996

A multi-substrate single-file model for ion-coupled transporters.

Ion-coupled transporters are simulated by a model that differs from contemporary alternating-access schemes. Beginning with concepts derived from multi-ion pores, the model assumes that substrates (both inorganic ions and small organic molecules) hop a) between the solutions and binding sites and b) between binding sites within a single-file pore. No two substrates can simultaneously occupy the same site. Rate constants for hopping can be increased both a) when substrates in two sites attract each other into a vacant site between them and b) when substrates in adjacent sites repel each other. Hopping rate constants for charged substrates are also modified by the membrane field. For a three-site model, simulated annealing yields parameters to fit steady-state measurements of flux coupling, transport-associated currents, and charge movements for the GABA transporter GAT1. The model then accounts for some GAT1 kinetic data as well. The model also yields parameters that describe the available data for the rat 5-HT transporter and for the rabbit Na(+)-glucose transporter. The simulations show that coupled fluxes and other aspects of ion transport can be explained by a model that includes local substrate-substrate interactions but no explicit global conformational changes.

Animals↗
PubMed1993

Guanidinium chloride induction of partial unfolding in amide proton exchange in RNase A.

Amide (NH) proton exchange rates were measured in 0.0 to 0.7 M guanidinium chloride (GdmCl) for 23 slowly exchanging peptide NH protons of ribonuclease A (RNase A) at pH* 5.5 (uncorrected pH measured in D2O), 34 degrees C. The purpose was to find out whether GdmCl induces exchange through binding to exchange intermediates that are partly or wholly unfolded. It was predicted that, when the logarithm of the exchange rate is plotted as a function of the molarity of GdmCl, the slope should be a measure of the amount of buried surface area exposed to GdmCl in the exchange intermediate. The results indicate that these concentrations of GdmCl do induce exchange by means of a partial unfolding mechanism for all 23 protons; this implies that exchange reactions can be used to study the unfolding and stability of local regions. Of the 23 protons, nine also show a second mechanism of exchange at lower concentrations of GdmCl, a mechanism that is nearly independent of GdmCl concentration and is termed "limited structural fluctuation."

Guanidine↗
PubMed1986

Long-range electron transfer in heme proteins.

Kinetic experiments have conclusively shown that electron transfer can take place over large distances (greater than 10 angstroms) through protein interiors. Current research focuses on the elucidation of the factors that determine the rates of long-range electron-transfer reactions in modified proteins and protein complexes. Factors receiving experimental and theoretical attention include the donor-acceptor distance, changes in geometry of the donor and acceptor upon electron transfer, and the thermodynamic driving force. Recent experimental work on heme proteins indicates that the electron-transfer rate falls off exponentially with donor-acceptor distance at long range. The rate is greatly enhanced in proteins in which the structural changes accompanying electron transfer are very small.

Animals↗
PubMedDate not supplied

Systemic immune response of young chickens orally immunized with bovine serum albumin.

The efficacy of oral immunization, with and without commercial adjuvants, to mount a systemic immune response in young chickens was studied. Bovine serum albumin (BSA) mixed with a pegylated C8/C10 mono/di-glyceride, (Softigen), or Cholera toxin B-subunit (CTB), administered orally by gavage to 15-day-old chickens resulted in circulating immunospecific anti-BSA IgG, IgM and IgA antibodies. Continuous 5-day oral administration of BSA without adjuvant also resulted in immunospecific IgM and IgA antibodies in the circulation of chickens first immunized at 15 days of age; and immunospecific antibodies of all three classes in chickens first immunized when they were 22 days old. IgG and IgM serum concentrations were more than 4 to 10 times higher, respectively, in CTB- and Softigen-treated chickens as compared to chickens immunized without adjuvants. The IgA response in the orally immunized chickens seemed unaffected by CTB and Softigen. The antibody concentrations in chickens immunized subcutaneously with BSA emulsified in Freund's Incomplete Adjuvant (FIA) were approximately 10 times higher than those of the chickens orally immunized using CTB and Softigen.

Administration, Oral↗