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L Mayne

Publications and source records attributed to L Mayne.

26 records · Page 2Linked to original sources
PubMed1994

Protein stability parameters measured by hydrogen exchange.

The hydrogen exchange (HX) rates of the slowest peptide group NH hydrogens in oxidized cytochrome c (equine) are controlled by the transient global unfolding equilibrium. These rates can be measured by one-dimensional nuclear magnetic resonance and used to determine the thermodynamic parameters of global unfolding at mild solution conditions well below the melting transition. The free energy for global unfolding measured by hydrogen exchange can differ from values found by standard denaturation methods, most notably due to the slow cis-trans isomerization of the prolyl peptide bond. This difference can be quantitatively calculated from basic principles. Even with these corrections, HX experiments at low denaturant concentration measure a free energy of protein stability that rises above the usual linear extrapolation from denaturation data, as predicted by the denaturant binding model of Tanford.

Amines↗
PubMed1994

The barriers in protein folding.

Elimination of an interaction which forms in denatured cytochrome c enables the majority of the molecules to fold to the native state on a 15 ms time scale, without populating observable intermediates. These results are contrary to the current view that particular steps in protein folding, including the supposedly rate-limiting molten globule to native transition, are intrinsically slow. Instead it appears that intermediates characterized so far may be kinetically trapped by barriers that are optional rather than integral to the folding process. Major barriers may result from misorganization of the chain in the initial condensation step.

Animals↗
PubMed1993

Primary structure effects on peptide group hydrogen exchange.

The rate of exchange of peptide group NH hydrogens with the hydrogens of aqueous solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo- and polypeptides and, surprisingly, confirmed. Reference rates for alanine-containing peptides were determined and effects of temperature considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured oligo- and polypeptides. The application of this approach to protein studies is discussed.

Alanine↗
PubMed1992

Effect of antibody binding on protein motions studied by hydrogen-exchange labeling and two-dimensional NMR.

We have used hydrogen-exchange labeling detected by 2D NMR to study antibody-protein interactions for two monoclonal antibodies raised against horse cytochrome c. The data show that these antibodies bind mainly to the large 37-59 omega-loop of the cytochrome c molecule. In addition, the results provide some suggestive evidence concerning units of local structural flexibility in cytochrome c.

Antibodies, Monoclonal↗
PubMed1992

Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR.

HX-labeling experiments in the pH-pulse mode show that protein folding can be remarkably fast. A near-native form can be reached within milliseconds. Experimental analysis of the folding process on the millisecond-to-second time scale depends upon the presence of kinetic barriers that avoid apparent two-step folding. A common barrier produces molecular intermediates; disparate barriers produce population heterogeneity that makes analysis more difficult. Results available exhibit an early, native-like two-helix intermediate in cytochrome c, an extensive, native-like, beta-sheet-plus-helix intermediate in RNase A, and a late native-like molten globular intermediate in barnase. These differences appear to reflect chance differences in the placement of the determining kinetic barriers. Requirements for observing kinetic folding intermediates are difficult to satisfy, so most intermediates are not seen, and intermediates that are seen often represent the sum of multiple preceding steps.

Deuterium↗
PubMed1990

Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts.

We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

Antigens, Polyomavirus Transforming↗
PubMed1979

Abnormal kinetics of DNA synthesis in ultraviolet light-irradiated cells from patients with Cockayne's syndrome.

Cells from patients with the hereditary disorder Cockayne's syndrome and from the sun-sensitive individual, 11961, are sensitive to the lethal effects of ultraviolet light (UV) but have no detectable defect in either excision- or postreplication repair after UV irradiation. In normal cells and in Cockayne heterozygotes, UV causes a depression in the rate of DNA-replicative synthesis followed by a recovery of normal rates 5 to 8 hr after irradiation. In Cockayne and 11961 cells, the initial depression in DNA synthesis is the same as that in normal cells, but no subsequent recovery is observed. The recovery of DNA synthesis in normal cells appears to be unaffected by fluorodeoxyuridine but inhibited by cycloheximide. This suggests a possible requirement for de novo protein synthesis, but there are a number of alternative interpretations of these data.

Cells, Cultured↗