Biomedical subjects

R E Burton

Publications and source records attributed to R E Burton.

11 recordsLinked to original sources

PubMed2001

Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine.

ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.

Adenosine Triphosphatases↗

PubMed2000

Dynamics of substrate denaturation and translocation by the ClpXP degradation machine.

ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2000

An evolutionary bridge to a new protein fold.

Arc repressor bearing the N11L substitution (Arc-N11L) is an evolutionary intermediate between the wild type protein, in which the region surrounding position 11 forms a beta-sheet, and a double mutant 'switch Arc', in which this region is helical. Here, Arc-N11L is shown to be able to adopt either the wild type or mutant conformations. Exchange between these structures occurs on the millisecond time scale in a dynamic equilibrium in which the relative populations of each fold depend on temperature, solvent conditions and ligand binding. The N11L mutation serves as an evolutionary bridge from the beta-sheet to the helical fold because in the mutant, Leu is an integral part of the hydrophobic core of the new structure but can also occupy a surface position in the wild type structure. Conversely, the polar Asn 11 side chain serves as a negative design element in wild type Arc because it cannot be incorporated into the core of the mutant fold.

Amino Acid Sequence↗

PubMed2000

Novel disulfide engineering in human carbonic anhydrase II using the PAIRWISE side-chain geometry database.

An analysis of the pairwise side-chain packing geometries of cysteine residues observed in high-resolution protein crystal structures indicates that cysteine pairs have pronounced orientational preferences due to the geometric constraints of disulfide bond formation. A potential function was generated from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase II (HCAII). Three double-cysteine variants of HCAII were purified and the effective concentrations of their thiol groups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stability were characterized by circular dichroism, enzymatic activity, sulfonamide binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disulfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, the effects of cysteine substitutions on the native state and the stabilization of compact nonnative states by the disulfide can override any stabilizing effect of the cross-link.

Carbonic Anhydrases↗

PubMed1998

Folding kinetics of a fluorescent variant of monomeric lambda repressor.

A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degreesC using fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation curves obtained by circular dichroism, fluorescence, and NMR are superimposable. Stopped-flow analysis indicates that in the absence of denaturants the folding reaction is complete within the dead-time of the experiment. Within higher denaturant conditions, where the folding rate is slower, NMR and stopped-flow agree on the folding and unfolding rates of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant folds within 2 ms. Extrapolation indicates that the folding time is 20 micro(s) in the absence of denaturants. All folding and unfolding reactions displayed monoexponential kinetics, and no burst-phases were observed. In addition, the thermodynamic parameters Delta G and meq obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state Dleft and right arrow N folding model.

Amino Acid Substitution↗

PubMed1998

Protein folding dynamics: quantitative comparison between theory and experiment.

The development of a quantitative kinetic scheme is a central goal in mechanistic studies of biological phenomena. For fast-folding proteins, which lack experimentally observable kinetic intermediates, a quantitative kinetic scheme describing the order and rate of events during folding has yet to be developed. In the present study, the folding mechanism of monomeric lambda repressor is described using the diffusion-collision model and estimates of intrinsic alpha-helix propensities. The model accurately predicts the folding rates of the wild-type protein and five of eight previously studied Ala left and right arrow Gly variants and suggests that the folding mechanism is distributed among multiple pathways that are highly sensitive to the amino acid sequence. For example, the model predicts that the wild-type protein folds through a small number of pathways with a folding time of 260 micros. However, the folding of a variant (G46A/G48A) is predicted to fold through a large number of pathways with a folding time of 12 micros. Both folding times quantitatively agree with the experimental values at 37 degrees C extrapolated to 0 M denaturant. The quantitative nature of the diffusion-collision model allows for rigorous experimental tests of the theory.

Bacteriophage lambda↗

PubMed1997

Analysis of the structure and stability of omega loop A replacements in yeast iso-1-cytochrome c.

Omega (omega)-loop A, residues 18-32 in wild-type yeast iso-1-cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low-temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native-like cytochrome c conformation, but are less stable than the wild-type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.

Amino Acid Sequence↗

PubMed1997

The energy landscape of a fast-folding protein mapped by Ala-->Gly substitutions.

A moderately stable protein with typical folding kinetics unfolds and refolds many times during its cellular lifetime. In monomeric lambda repressor this process is extremely rapid, with an average folded state lifetime of only 30 milliseconds. A thermostable variant of this protein (G46A/G48A) unfolds with the wild-type rate, but it folds in approximately 20 microseconds making it the fastest-folding protein yet observed. The effects of alanine to glycine substitutions on the folding and unfolding rate constants of the G46A/G48A variant, measured by dynamic NMR spectroscopy, indicate that the transition state is an ensemble comprised of a disperse range of conformations. This structural diversity in the transition state is consistent with the idea that folding chains are directed towards the native state by a smooth funnel-like conformational energy landscape. The kinetic data for the folding of monomeric lambda repressor can be understood by merging the new energy landscape view of folding with traditional models. This hybrid model incorporates the conformational diversity of denatured and transition state ensembles, a transition state activation energy, and the importance of intrinsic helical stabilities.

Alanine↗

PubMed1996

Microsecond protein folding through a compact transition state.

Dynamic NMR methods have been employed to measure the folding and unfolding rate constants of two extremely fast-folding proteins. lambda 6-85, a truncated, monomeric form of the N-terminal domain of lambda repressor, refolds with a lifetime of approximately 250 microseconds. These methods have also been applied to a thermostable lambda 6-85 variant with alanine substituted for glycine residues 46 and 48 in the third helix (G46A/G48A). Both proteins exhibit linear ln (kf,u) versus [urea] plots, consistent with two-state folding for both proteins. When extrapolated to 0M urea, the data indicate that G46A/G48A folds with a lifetime of less than 20 microseconds. The slopes of the ln (kf,u) versus [urea] curves (mu and mf) indicate that the modest Gly-->Ala double mutation dramatically changes the transition state solvent accessibility. The transition state for lambda 6-85 has a fractional accessibility (mu/(mu-mf)) of 0.61, whereas the transition state for G46A/G48A is much more native-like, with a fractional accessibility of 0.16. The extraordinary change in the folding pathway that these mutations induce suggests that the intrinsic stability of helix 3 is an important determinant of the folding mechanism.

DNA-Binding Proteins↗

PubMed1995

Experimental tests for protrusion and undulation pressures in phospholipid bilayers.

Theoretical treatments predict that strong entropic pressures between adjacent bilayer membranes can arise from out of plane motions caused by either thermally induced bending undulations of the entire bilayer [Harbich, W., & Helfrich, W. (1984) Chem. Phys. Lipids 36, 39-63; Evans, E. A., & Parsegian, V. A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7132-7136] or protrusions of individual lipid molecules from the bilayer surface [Israelachvili, J. N., & Wennerström, H. (1992) J. Phys. Chem. 96, 520-531]. To determine the relative contributions of these motions to the repulsive pressure between phospholipid bilayers, the osmotic stress/X-ray diffraction method was used to measure the range and magnitude of the total repulsive pressure, and micropipet methods were used to measure the bending moduli of phosphatidylcholine bilayers containing lysophosphatidylcholine and polyunsaturated diarachidonoylphosphatidylcholine (DAPC) bilayers. In the gel phase, incorporation of equimolar lysophosphatidylcholine into phosphatidylcholine bilayers caused the hydrocarbon chains from apposing monolayers to interdigitate, but did not appreciably change the equilibrium fluid spacing in excess buffer from its control value of 12 A. In contrast, the incorporation of equimolar lysophosphatidylcholine into liquid-crystalline phase phosphatidylcholine bilayers markedly increased the range of the repulsive pressure so that equilibrium fluid separation increased from 15 to 28 A, and also decreased the bilayer bending modulus from 5.1 x 10(-13) to 1.3 x 10(-13) erg. Liquid-crystalline DAPC bilayers had intermediate values of both equilibrium fluid separation (20 A) and bending modulus (2.8 x 10(-13) erg). Analysis of these data indicates that (1) the relative importance of entropic pressures compared to the hydration pressure depends strongly on the composition and structure of the bilayer, (2) the protrusion pressure may contribute to the total repulsive pressure at large pressures or small fluid spacings, and (3) the repulsive undulation pressure, together with the attractive van der Waals pressure, is a primary factor in determining the fluid spacing at low and/or zero applied pressures in liquid-crystalline bilayers.

Electrons↗

PubMed1993

The structure and function of omega loop A replacements in cytochrome c.

The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C. This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.

Amino Acid Sequence↗