Biomedical subjects

M Bycroft

Publications and source records attributed to M Bycroft.

51 records · Page 3Linked to original sources

PubMed1993

Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen-deuterium exchange.

The extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was characterized by comparing the differences in the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase. Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY-HMQC and TOCSY-HMQC spectra of a complex that had been prepared with uniformly 15N-labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of [15N]HMQC spectra of a sample prepared in the same manner exchanged into D2O. The largest changes in either chemical shift or hydrogen-deuterium exchange rate are observed for residues located in the active-site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site.

Bacterial Proteins↗

PubMed1993

Refolding of barnase mutants and pro-barnase in the presence and absence of GroEL.

Three mutants of barnase and a pro-barnase variant, which have a variety of different physical properties but the same overall protein structure, were analysed for their folding in the presence of the molecular chaperone GroEL. Mutants were chosen on the basis that changes in their refolding rate constants in solution are not correlated with the changes in their stability. All barnase variants fold considerably more slowly when bound to GroEL. However, barnase refolding on GroEL parallels that in solution: there is a linear relationship between the refolding rate constants, obtained for wild-type and all mutants of barnase, in the presence and absence of GroEL. Barnase is synthesized in vivo with a 13 amino acid pro-sequence attached to the N-terminus. The pro-sequence of pro-barnase is shown by NMR spectroscopy to be devoid of defined structure. The presence of this pro-sequence has no effect on the overall refolding rate constant or the activity of barnase. In the presence of GroEL, the refolding of pro-barnase is retarded relatively more strongly than that of wild-type and the mutant barnase proteins, suggesting that the pro-sequence provides additional binding sites for the chaperone.

Amino Acid Sequence↗

PubMed1992

The folding of an enzyme. V. H/2H exchange-nuclear magnetic resonance studies on the folding pathway of barnase: complementarity to and agreement with protein engineering studies.

Two major methods are currently being used to characterize transient intermediates during protein folding at the level of individual residues. Nuclear magnetic resonance (n.m.r.) measurements on the protection of peptide NH hydrogens against exchange with solvent during refolding can provide information about secondary structure formation. Protein engineering and kinetics can provide direct information about intramolecular interactions of protein side-chains and indirect evidence on secondary structure. These procedures have provided the most complete pictures so far about protein folding intermediates. Both methods have been applied to the characterization of an intermediate in the refolding of barnase. Although the two methods give complementary information, there are some regions of the protein where the methods overlap well. We show that, with one possible exception that is obscure, n.m.r. and protein engineering give identical results for those interactions that can be analysed by both methods. This suggests that these are valid approaches for the study of protein folding intermediates in the case of barnase and that the combination of the methods is a powerful analytical procedure. Information provided by n.m.r. data that is complementary to the protein engineering experiments is: (1) early formation of the C terminus of helix2; (2) early formation of helix3; (3) early formation of several beta-turns (46-49, 101-104 in loop5); and (5) partial formation of loop5. Confirmatory evidence of protein engineering data on the intermediate is: (1) helix1 is complete from residues 10 to 18; (2) the interactions between all beta-strands are present; (3) part of loop2 is not formed; (4) part of loop3 is formed; and (5) some specific tertiary interactions are not made. For some interactions the protein engineering and H/2H exchange methods overlap directly. The information obtained for direct overlap is self consistent.

Amino Acid Sequence↗

PubMed1991

Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis and NMR.

Phosphate is a competitive inhibitor of transesterification of GpC by the ribonuclease barnase. Barnase is significantly stabilized in the presence of phosphate against urea denaturation. The data are consistent with the existence of a single phosphate binding site in barnase with a dissociation constant, Kd, of 1.3 mM. The 2D 1H NMR spectrum of wild-type barnase with bound phosphate is assigned. Changes in chemical shifts and NOEs for wild type with bound phosphate compared with free wild type indicate that phosphate binds in the active site and that only small conformational changes occur on binding. Site-directed mutagenesis of the active site residues His-102, Lys-27, and Arg-87 to Ala increases the magnitude of Kd for phosphate by more than 20-fold. The 2D 1H NMR spectra of the mutants His-102----Ala, Lys-27----Ala, and Arg-87----Ala are assigned. Comparison with the spectra of wild-type barnase reveals that His-102----Ala and Lys-27----Ala have essentially the same structure as weild type, while some structural changes occur in Arg-87----Ala. It appears that phosphate binding by barnase is effected mainly by positively charge residues including His-102, Lys-27, and Arg-87. This may have applications for the design of phosphate binding sites in other proteins.

Amino Acid Sequence↗

PubMed1991

Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spectroscopy.

The solution conformation of the ribonuclease barnase has been determined by using 1H nuclear magnetic resonance (NMR) spectroscopy. The 20 structures were calculated by using 853 interproton distance restraints obtained from analyses of two-dimensional nuclear Overhauser spectra, 72 phi and 53 chi 1 torsion angle restraints, and 17 hydrogen-bond distance restraints. The calculated structures contain two alpha-helices (residues 6-18 and 26-34) and a five-stranded antiparallel beta-sheet (residues 50-55, 70-75, 85-91, 94-101, and 105-108). The core of the protein is formed by the packing of one of the alpha-helices (residues 6-18) onto the beta-sheet. The average RMS deviation between the calculated structures and the mean structure is 1.11 A for the backbone atoms and 1.75 A for all atoms. The protein is least well-defined in the N-terminal region and in three large loops. When these regions are excluded, the average RMS deviation between the calculated structures and the mean structure for residues 5-34, 50-56, 71-76, 85-109 is 0.62 A for the backbone atoms and 1.0 A for all atoms. The NMR-derived structure has been compared with the crystal structure of barnase [Mauguen et al. (1982) Nature (London) 297, 162-164].

Amino Acid Sequence↗

PubMed1991

Surface electrostatic interactions contribute little of stability of barnase.

Electrostatic interactions are believed to play an important role in stabilizing the native structure of proteins. We have quantified the contribution to stability of an interaction between two oppositely charged side-chains on the surface of barnase. Using site-directed mutagenesis, glutamate 28 and lysine 32 were introduced onto the solvent-accessible side of the second alpha-helix in barnase. These two residues are separated by one turn of the helix, and so are ideally situated for their opposite charges to interact. Double mutant cycle analysis reveals that the interaction between Glu28 and Lys32 contributes only approximately 0.2 kcal/mol to stability of the protein. All other interactions between exposed charged side-chains in barnase examined so far also contribute little to stability. We explain this low value by their location on the surface, rather than in the interior, of the protein.

Amino Acid Sequence↗

PubMed1991

Pathway and stability of protein folding.

We describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. The strategy is based on two steps. First, protein engineering is used to remove interactions that stabilize defined positions in barnase, the RNAse from Bacillus amyloliquefaciens. The consequent changes in stability are measured from the changes in free energy of unfolding of the protein. Second, each mutation is used as a probe of the structure around the wild-type side chain during the folding process. Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. The kinetic and thermodynamic data are combined and analysed to show the role of individual side chains in the stabilization of the folded, transition and intermediate states of the protein. The protein engineering experiments are corroborated by nuclear magnetic resonance studies of hydrogen exchange during the folding process. Folding is a multiphasic process in which alpha-helices and beta-sheet are formed relatively early. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining. The final steps involve the forming of loops and the capping of the N-termini of helices.

Amino Acid Sequence↗

PubMed1991

Aromatic-aromatic interactions and protein stability. Investigation by double-mutant cycles.

The side-chains of phenylalanine and tyrosine residues in proteins are frequently found to be involved in pairwise interactions. These occur both within repeating elements of secondary structure and in tertiary and quaternary interactions. It has been suggested that they are important in protein folding and stability, and non-bonded potential energy calculations indicate that a typical aromatic-aromatic interaction has an energy of between -1 and -2 kcal/mol and contributes between -0.6 and -1.3 kcal/mol to protein stability. There is such an aromatic pair on the solvent-exposed face of the first alpha-helix of barnase, the small ribonuclease from Bacillus amyloliquefaciens. The edge of the aromatic ring of Tyr17 interacts with the face of that of Tyr13. The two residues have been mutated both singly and pairwise to alanine, and their free energies of unfolding determined by denaturation with urea. Application of the double-mutant cycle analysis gives an interaction energy of -1.3 kcal/mol for the aromatic pair in the folded protein relative to solvation by water in the unfolded protein. This value is similar to that calculated from the change in surface-accessible area between the rings on the formation of the pair. Analysis of a further double-mutant cycle in which the Tyr residues are mutated to Phe indicates that the aromatic-aromatic interactions of Tyr/Tyr and Phe/Phe make identical contributions to protein stability. However, Tyr is preferred to Phe by 0.3(+/- 0.04) kcal/mol at the solvent-exposed face of the alpha-helix.

Amino Acid Sequence↗

PubMed1990

Strength and co-operativity of contributions of surface salt bridges to protein stability.

Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.

Alanine↗

PubMed1990

Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles.

Coulombic interactions between charges on the surface of proteins contribute to stability. It is difficult, however, to estimate their importance by protein engineering methods because mutation of one residue in an ion pair alters the energetics of many interactions in addition to the coulombic energy between the two components. We have estimated the interaction energy between two charged residues, Asp-12 and Arg-16, in an alpha-helix on the surface of a barnase mutant by invoking a double-mutant cycle involving wild-type enzyme (Asp-12, Thr-16), the single mutants Thr----Arg-16 and Asp----Ala-12, and the double mutant Asp----Ala-12, Thr----Arg-16. The changes in free energy of unfolding of the single mutants are not additive because of the coulombic interaction energy. Additivity is restored at high concentrations of salt that shield electrostatic interactions. The geometry of the ion pair in the mutant was assumed to be the same as that in the highly homologous ribonuclease from Bacillus intermedius, binase, which has Asp-12 and Arg-16 in the native enzyme. The ion pair does not form a hydrogen-bonded salt bridge, but the charges are separated by 5-6 A. The mutant barnase containing the ion pair Asp-12/Arg-16 is more stable than wild type by 0.5 kcal/mol, but only a part of the increased stability is attributable to the electrostatic interaction. We present a formal analysis of how double-mutant cycles can be used to measure the energetics of pairwise interactions.

Amino Acid Sequence↗

PubMed1990

Sequential assignment of the 1H nuclear magnetic resonance spectrum of barnase.

Two-dimensional nuclear magnetic resonance spectroscopy has been used to study the bacterial ribonuclease barnase (MW 12,382). Resonance assignments have been made for protons in all of the 110 residues. Analysis of medium- and long-range contacts in NOESY spectra has demonstrated that the major elements of secondary structure in barnase in solution are essentially identical with those found in the crystal structure.

Amino Acid Sequence↗

PubMed1990

Transient folding intermediates characterized by protein engineering.

Kinetic experiments on engineered mutants of barnase detect an intermediate on the folding pathway and allow the mapping of the tertiary interactions of the side chains and their energetics. Many of the interactions present in the final folded state tend to be either fully formed or not formed at all in the intermediate or subsequent transition state for folding, but the hydrophobic core becomes progressively consolidated. These methods in combination with NMR provide extensive structural characterization of the folding intermediate and the sequence of events in the folding pathway.

Bacillus↗

PubMed1990

Detection and characterization of a folding intermediate in barnase by NMR.

Protein engineering is being developed for mapping the energetics and pathway of protein folding. From kinetic studies on wild-type and mutant proteins, the sequence and energetics of formation of tertiary interactions of side chains can be mapped and the formation of secondary structure inferred. Here we cross-check and complement results from this approach by using a recently developed procedure that traps and characterizes secondary structure in intermediate states using 1H NMR. The refolding of barnase is shown to be a multiphasic process in which the secondary structure in alpha-helices and beta-sheets and some turns is formed more rapidly than is the overall folding.

Amino Acid Sequence↗

PubMed1988

Stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain.

The alpha-helix in proteins has a dipole moment resulting from the alignment of dipoles of the peptide bond which can perturb the pKas of ionizing groups. One of the two histidine residues (His18) in barnase, the small ribonuclease from Bacillus amyloliquefaciens, is located at the negatively charged end (C-terminal) of an alpha-helix. From NMR titrations of wild-type and engineered mutants we find that the pKa of His18 is 7.9 in wild-type enzyme, 1.6 units above the value in the urea-denatured enzyme and in model peptides. This implies that there is a favourable interaction between the protonated form of His18 and the alpha-helix that should stabilize the native structure at neutral pH by 2.1 kcal mol-1. Denaturation at various values of pH of wild-type and muant enzymes engineered at position 18 shows that this is so. The increase in stability of the enzyme as the pH changes from 8.5 to 6.3 is attributable to this interaction, and the pH-stability curve fits pKa values for His18 in native and urea-denatured enzymes that are consistent with the NMR data.

Bacterial Proteins↗

PubMed1988

Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis.

A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKas for the six histidines in this enzyme. The pKas of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.

Bacillus↗