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A Matouschek

Publications and source records attributed to A Matouschek.

27 records · Page 2Linked to original sources
PubMed1992

The folding of an enzyme. IV. Structure of an intermediate in the refolding of barnase analysed by a protein engineering procedure.

The pathway of refolding of barnase has been analysed by the protein engineering method using phi plots. The description comprises a folding intermediate, a major transition state (the unfolding transition state) and the fully folded structure. Over 40 mutations have been analysed in the different structural motifs, frequently with several probes in each region. Many of the mutations in this study give phi values for formation of the intermediate of 0, showing that the relevant regions of the structure are as fully unfolded in the intermediate as the unfolded state. Some folding phi values are close to unity, indicating that those regions are fully formed in the intermediate. Even if the data do not report back on a single intermediate but give the averaged properties of a heterogeneous population of sequential or parallel intermediates, then this simplicity of phi data shows that the intermediates tend to have structural features in common. Many phi values are intermediate between those for the unfolded state and the transition state, consistent with either partial structure formation in a single intermediate or a heterogeneous mixture of populations, although the former is more likely. The data are consistent with the intermediate, or collection of intermediates, being on the reaction pathway, rather than side products, because the phi values increase throughout the folding pathway. The main conclusions on the formation of substructure and sequence of folding events from the phi plots are as follows. (1) The major hydrophobic core (core1) begins to form in the intermediate and strengthens in the major transition state. The centre of the core is formed earlier and is stronger in the intermediate and in the transition state than are the edges. (2) Core2 is not formed until after the major transition state. (3) Core3 begins to form in the intermediate and is compact in the transition state. (4) Loop2, loop4 and part of loop1 do not fold until after the major transition state, but the guanosine-binding loop (loop3) is formed in the intermediate and loop5 is partially formed in the intermediate and the transition state. (5) The centre of the beta-sheet is substantially formed in the intermediate, and is fully present in the transition state, but the edges, and associated turns, are definitely weakened.(ABSTRACT TRUNCATED AT 400 WORDS)

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↗
PubMed1992

The folding of an enzyme. VI. The folding pathway of barnase: comparison with theoretical models.

The sequence of events in the refolding pathway of barnase has been analysed to search for general principles in protein folding. There appears to be a correlation between burying hydrophobic surface area and early folding events. All the regions that fold early interact extensively with the beta-sheet. These interactions involve predominantly hydrophobic interactions and the burial of very extensive hydrophobic areas in which multiple, close, hydrophobic-hydrophobic contacts are established around a central group of aliphatic residues. There is no burial of hydrophilic residues in these regions; those that are partly screened from the solvent make hydrogen bonds. All the regions or interactions that are made late in the folding pathway do not make extensive contacts with the beta-sheet. Their buried hydrophobic regions lack a central hydrophobic residue or residues around which other hydrophobic residues pack. Further, in some of these regions there is an extensive burial of hydrophilic residues. The results are consistent with one of the earlier events in protein folding being the local formation of native-like secondary structure elements driven by local hydrophobic surface burial. A possible candidate for an initiation site is a beta-hairpin between beta-strands 3 and 4 that is conserved in the microbial ribonuclease family. A comparison of structures in this family shows that those regions that can be superimposed, or have sequence homology, correspond to elements of structure that are formed and interact with each other early in the folding pathway, suggesting that some of these residues could be involved in directing the folding process. The data on barnase combined with results from other laboratories suggest the following tentative conclusions for the refolding of small monomeric proteins. (1) The refolding pathway is, at least in part, sequential and of compulsory order. (2) Secondary structure formation is driven by local hydrophobic surface burial and precedes the formation of most tertiary interactions. These elements are then stabilized and sometimes elongated by tertiary interactions. It is plausible that there are stop signals encoded in the linear sequence that prevent the elongation of isolated secondary structure elements in solution to a larger extent than is found in the folded protein. (3) Many tertiary interactions are not very constrained in the intermediate but become more and more defined as the hydrophobic cores consolidate, loop structures form and the configuration of surface residues takes place. The interactions between different elements of secondary structure are the last ones to be consolidated while the interactions within the secondary structure elements are consolidated earlier.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Pathway of protein folding.

The order of formation of substructures in the folding of barnase has been determined by a protein engineering procedure and corroborated and complemented by NMR experiments. Early events are the formation of the centre of the beta-sheet and the C-terminus of the major alpha-helix. These later dock to form the major hydrophobic core. Structural studies of fragments of barnase in solution show that a peptide that spans the major alpha-helix is found to contain a significant fraction of its C-terminal region in the helical structures. The formation of the native secondary structure as an early event in folding and in isolated fragments is accompanied by considerable burial of hydrophobic surfaces. The experimental data support a model for protein folding in which initiation sites in secondary structure are driven by local hydrophobic interactions, and their docking via further hydrophobic interactions drives the formation of tertiary structure.

Bacterial Proteins↗
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↗
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↗