Biomedical subjects

R O Fox

Publications and source records attributed to R O Fox.

44 records · Page 3Linked to original sources

PubMed1989

Transfer of a beta-turn structure to a new protein context.

Four-residue beta-turns and larger loop structures represent a significant fraction of globular protein surfaces and play an important role in determining the conformation and specificity of enzyme active sites and antibody-combining sites. Turns are an attractive starting point to develop protein design methods, as they involve a small number of consecutive residues, adopt a limited number of defined conformations and are minimally constrained by packing interactions with the remainder of the protein. The ability to substitute one beta-turn geometry for another will extend protein engineering beyond the redecoration of fixed backbone conformations to include local restructuring and the repositioning of surface side chains. To determine the feasibility and to examine the effect of such a structural modification on the fold and thermodynamic stability of a globular protein, we have substituted a five-residue turn sequence from concanavalin A for a type I' beta-turn in staphylococcal nuclease. The resulting hybrid protein is folded and has full nuclease enzymatic activity but reduced thermodynamic stability. The crystal structure of the hybrid protein reveals that the guest turn sequence retains the conformation of the parent concanavalin A structure when substituted in the nuclease host.

Binding Sites↗

PubMed1989

A magnetization-transfer nuclear magnetic resonance study of the folding of staphylococcal nuclease.

The equilibrium between alternative folded states of a globular protein, staphylococcal nuclease, has been investigated by using 1H NMR. Magnetization-transfer experiments have revealed the existence of a related structural heterogeneity of the unfolded state, and quantitative analysis of a series of these experiments has permitted the kinetics of folding and interconversion of the different states to be explored. A model based on cis/trans isomerism at the peptide bond preceding Pro-117 has been developed to account for the results. This model, recently supported by a protein-engineering experiment [Evans et al. (1987) Nature (London) 329, 266], has been used to interpret the kinetic data, providing insight into the nature of the folding processes. The predominance of the cis-proline form in the folded state is shown to derive from a large favorable enthalpy term resulting from more effective overall folding interactions. The kinetics of folding and isomerization are shown to occur on similar time scales, such that more than one pathway between two states may be significant. It has been possible, however, to compare the direct folding and unfolding rates within the cis- and trans-proline-containing populations, with results suggesting that the specific stabilization of the cis peptide bond is effective only at a late stage in the folding process.

Isomerism↗

PubMed1989

Fluorescence lifetime studies with staphylococcal nuclease and its site-directed mutant. Test of the hypothesis that proline isomerism is the basis for nonexponential decays.

Using frequency domain methods, the fluorescence decay of Trp-140 in staphylococcal nuclease and its site-directed mutant (Pro-117----Gly) has been examined. Based on nuclear magnetic resonance (NMR) studies (Evans, P. A., C. M. Dobson, R. A. Kautz, G. Hatfull, and R. O. Fox. 1987. Nature [Lond.]. 329:266-268), it is believed that nuclease exists in two macroscopic, native conformations and that the slow interconversion of these conformations is controlled by the cis----trans isomerization of Pro-117. The above mutant shows only one native conformation in NMR experiments. To test the hypothesis that the biexponential fluorescence decay of Trp-140 of nuclease can also be related to the existence of these conformational states of the protein, we have compared the decay patterns of the wild type and mutant. Essentially no difference was observed, which indicates that there is some other basis for the nonexponential decay of Trp-140. We have used global nonlinear least squares analysis to link the fit of data at several temperatures.

Binding Sites↗

PubMed1988

Crystallization of an anti-2,2,6,6-tetramethyl-1-piperidinyloxy-dinitrophenyl monoclonal antibody Fab fragment with and without bound hapten.

The Fab fragment of a monoclonal antibody, AN02, specific for a 2,2,6,6-tetramethyl-1-piperidinyloxy-dinitrophenyl hapten was crystallized both with and without bound hapten. Both crystals formed in phosphate-buffered saline (150 mM-NaCl, 10 mM-Na2PO4, 0.02% (w/v) NaN3 (pH 7.4) at 4 degrees C and diffracted beyond 2.2 A resolution (1A = 0.1 nm). Fab with bound hapten crystallizes in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 73.23 A, c = 373.8 A, alpha = beta = 90 degrees and gamma = 120 degrees. Unoccupied Fab also crystallizes in space group P6(1)22 or P6(5)22 with cell dimensions a = b = 73 A, c = 377 A, alpha = beta = 90 degrees and gamma = 120 degrees.

Antibodies, Monoclonal↗

PubMed1982

A voltage-gated ion channel model inferred from the crystal structure of alamethicin at 1.5-A resolution.

The crystal structure of alamethicin in nonaqueous solvent has been determined, and refined at 1.5-A resolution. The molecular conformation of the three crystallographically independent molecules is largely alpha-helical with a bend in the helix axis at an internal proline residue. The helix structure is highly amphipathic as most of the solvent-accessible polar atoms lie on a narrow strip of surface parallel to the helix axis. Molecular models for the voltage-gated ion channel, with n-fold symmetry and based on the molecular conformations observed in the crystal, are characterized by strong surface complementarity, a hydrophilic interior and a hydrophobic exterior. The channel structures are stabilized by a hydrated annulus of hydrogen-bonded glutamine residues which produce the greatest restriction in the channel diameter.

Alamethicin↗

PubMedDate not supplied

Recombinant antibodies possessing novel effector functions.

The introduction into lymphocytes of immunoglobulin-gene DNA that has been manipulated in vitro allows the production of novel antibodies. In this way, cell lines have been established that secrete hapten-specific antibodies in which the Fc portion has been replaced either with an active enzyme moiety or with polypeptide displaying c-myc antigenic determinants.

Animals↗

PubMedDate not supplied

Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy.

It is generally accepted that a globular protein in its native state adopts a single, well-defined conformation. However, there have been several reports that some proteins may exist in more than one distinct folded form in equilibrium. In the case of staphylococcal nuclease, evidence for multiple conformations has come from electrophoretic and NMR studies, although there has been some controversy as to whether these are actually interconvertible forms of the same molecular species. Recently, magnetization transfer (MT)-NMR has been developed as a means of studying the kinetics of conformational transitions in proteins. In the study reported here, this approach has been extended and used to demonstrate the presence of at least two native forms of nuclease in equilibrium and to study their interconversion with the unfolded state under the conditions of the thermal unfolding transition. The experiments reveal that two distinct native forms of the protein fold and unfold independently and that these can interconvert directly as well as via the unfolded state. The spectra of the different forms suggest that they are structurally similar but the MT experiments show that the kinetics of folding and unfolding are quite different. Characterization of this behaviour will, therefore, have important implications for our understanding of the relationship between structure and folding kinetics.

Magnetic Resonance Spectroscopy↗

PubMedDate not supplied

Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis.

Nuclear magnetic resonance (NMR) studies have shown that two distinct folded conformations of staphylococcal nuclease coexist in solution and that these two states can interconvert directly without passing through an unfolded state. These experiments have also revealed that the two forms have very different folding kinetics, although the possibility that one component is an obligatory intermediate for the folding of the other form could be discounted. Here we report NMR data which show that alternative unfolded states are also distinguishable. These observations led us to hypothesize that cis/trans isomerism at a single peptide bond between a proline and its preceding residue might be the origin of the conformational multiplicity. Proline 117 was identified as a likely candidate for the site concerned and a mutant protein, in which Pro 117 was replaced by Gly, was constructed in order to test this. Alternative conformations are not observed in the spectrum of this mutant, lending powerful support to this hypothesis.

Isomerism↗