NMR Studies of the unfolding of ribonuclease by guanidine hydrochloride. Evidence for intermediate states

Benz, Frederick W.; Roberts, Gordon C. K.

doi:10.1016/0014-5793(73)80034-1

Cited by 14 publications

(2 citation statements)

References 17 publications

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“…Dill and Shortle (1991) have proposed continuous change between the N-and U-states as a general model for protein denaturation. Also, NMR observations on some proteins during denaturant titrations have shown that changes in the outer parts of the molecule proceed before the unfolding of the hydrophobic core (Bradburg & King, 1969;Benz & Roberts, 1973). More recently, an intermediate state which expands continuously with increasing concentration of GdnHCl has been observed in the case of the heat shock protein Dnak (Polleros et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

Motional Dynamics of a Buried Tryptophan Reveals the Presence of Partially Structured Forms during Denaturation of Barstar

et al. 1996

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A double mutant of the single-domain protein barstar having a single tryptophan (W53) was made by mutating the remaining two tryptophans (W38 and W44) into phenylalanines. W53 is buried in the core of barstar. Time-resolved fluorescence of the mutant barstar (W38FW44F) showed that W53 has a single fluorescence lifetime in the native (N) state and has three lifetimes in the molten globule-like low-pH (A) form. Quenching of fluorescence by either KI or acrylamide showed that W53 is solvent inaccessible in the N-state and fairly accessible in the A-form. The denaturation of W38FW44F by guanidine hydrochloride (GdnHCI) was monitored by several probes: near-UV and far-UV circular dichroism (CD), fluorescence intensity, and steady-state and time-resolved fluorescence anisotropy. While the unfolding transitions observed through CD and fluorescence intensity coincided with each other (midpoint approximately 1.8 M GdnHCI), the transition observed through the steady-state fluorescence anisotropy was markedly different from others. Initially, the anisotropy increased with the increase in the concentration of GdnHCI and decreased subsequently. The midpoint of this titration was 2.2 M GdnHCI. Picosecond time-resolved fluorescence anisotropy showed that W38FW44F has a single rotational correlation time of 4.1 ns in the native (N) state and 1.5 as in the unfoled (U) state (6 M GdnHCI). These could be explained as being due to the absence of motional freedom of W53 in the N-state and the presence of rotational freedom in the U-state. In the intermediate concentration region (1.8-3.0 M GdnHCI), the anisotropy decays showed at least two coorrelation times, approximately 1 and 6-12 ns. These two correlation times are ascribed to partially structured forms leading to hindered rotation of W53. Thus, the usefulness of time-resolved fluorescence anisotropy in detecting partially folded structures is demonstrated.

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Section: Discussionmentioning

confidence: 99%

Motional Dynamics of a Buried Tryptophan Reveals the Presence of Partially Structured Forms during Denaturation of Barstar

et al. 1996

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“…This led naturally to the concept of nucleation centres -regions, perhaps, of a-helix or /?-pleated sheet -to direct the folding process, an idea that is receiving growing experimental support (Anfinsen 19735 W etlaufer 1973) For example, intermediates have been detected and tentatively identified in n.m.r. studies of the unfolding of staphylococcal nuclease (Jardetzky et al 1972) and ribonuclease-A (Benz & Roberts 1973) caused by a variety of denaturing agents. Intermediates have also been recognized in the folding of ribonuclease (Tsong, Baldwin & Elson 1972), lysozyme (Wetlaufer & Ristow 1973), cytochrome c (Ikai, Fish & Tanford 1973) and pancreatic trypsin inhibitor (Creighton 1974); and this list is by no means exhaustive.…”

Section: Monomeric Proteinsmentioning

confidence: 99%

Self-assembly of biological macromolecules

Perham

1975

Phil. Trans. R. Soc. Lond. B

103

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The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.

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The effect of chloroplatinite on the 1H NMR spectrum of ribonuclease Solvent accessibility of methionine residues

Sadler¹,

Benz²,

Roberts³

1974

Biochimica et Biophysica Acta (BBA) - Protein Structure

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NMR Studies of the unfolding of ribonuclease by guanidine hydrochloride. Evidence for intermediate states

Cited by 14 publications

References 17 publications

Motional Dynamics of a Buried Tryptophan Reveals the Presence of Partially Structured Forms during Denaturation of Barstar

Motional Dynamics of a Buried Tryptophan Reveals the Presence of Partially Structured Forms during Denaturation of Barstar

Self-assembly of biological macromolecules

The effect of chloroplatinite on the 1H NMR spectrum of ribonuclease Solvent accessibility of methionine residues

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