Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped‐flow circular dichroism

Kalnin, N.N.; Kuwajima, Kunihiro

doi:10.1002/prot.340230206

Cited by 26 publications

(34 citation statements)

References 47 publications

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“…The global rates of 5 sec −1 and 0.13 sec −1 observed in our time‐resolved labeling experiments are consistent with similar rates detected by other methods (Sugawara et al 1991; Chen and Tsong 1994; Kalnin and Kuwajima 1995; Maki et al 1999; Walkenhorst et al 1997). There appears to be no detectable protection of amides associated with a 1‐sec −1 phase observed by stopped flow, which is consistent with our assignment of this phase to a nonproline cis ‐ trans isomerism in our previous stopped‐flow fluorescence studies (Walkenhorst et al 1997).…”

Section: Discussionsupporting

confidence: 91%

“…More recently, several lines of study, including calorimetry (Carra et al 1994; Carra and Privalov 1995 Carra and Privalov 1996), NMR (Wang and Shortle 1995; Gillespie and Shortle 1997), pulsed hydrogen exchange (Jacobs and Fox 1994), mutational studies (Kalnin and Kuwajima 1995), and kinetic analysis of a proline‐free (Pro − ) SNase variant (Walkenhorst et al 1997) have converged to indicate that the β‐sheet subdomain of the protein is the most stable to denaturation and among the first regions of the protein to fold. By coupling hydrogen exchange labeling techniques with NMR detection of individual amide protons, it is possible to localize folding events to specific regions of the protein (Roder and Wüthrich 1986; Roder et al 1988; Udgaonkar and Baldwin 1988).…”

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confidence: 99%

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Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

et al. 2002

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Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid-denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly 15 N-labeled H124L SNase was monitored as a function of refolding time by heteronuclear two-dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen-bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the ␤-barrel and to a lesser degree in the ␣-helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a ␤-hairpin formed by strands 2 and 3 of the ␤-barrel domain and a single site near the C-terminus. The results indicate that formation of stable hydrogen-bonded structure in a core region of the ␤-sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.Keywords: Staphylococcal nuclease; pulse labeling; hydrogen exchange; NMR; protein folding Staphylococcal nuclease (SNase) has long been recognized as an excellent model for studies of protein folding and stability (Anfinsen et al. 1972;. Much insight has been gained, for example, about the role of proline isomerism in protein folding from studies on SNase (Evans et al. 1987(Evans et al. , 1989Kuwajima et al. 1991;Hinck et al. 1996;Maki et al. 1999) and other model proteins (Brandts et al. 1975;Ernst et al. 1985;Kiefhaber et al. 1990;Herning et al. 1991;Schmid 1993). SNase has also been a source of information on the contribution of cis-trans isomerism of prolines to protein conformational heterogeneity and on the interplay between local conformation and global stability as studied by the effects of single-site mutations (Alexandrescu et al. 1990). In particular, the presence of a cis peptide bond at position 116-117 in SNase (see Fig. 1) has been found to strongly increase the stability of SNase (Hinck et al. 1996). Although the structure of SNase can be divided into two subdomains (Fig. 1), an N-terminal ␤-barrel domain comprised of five antiparallel ␤-strands and a C-ter- ; HSMQC, heteronuclear single-quantum, multiple-quantum correlation; pdTp, deoxythymidine-3Ј,5Ј-bisphosphate; pH*, pH of a sample dissolved in D 2 O as determined by an uncorrected glass electrode measurement; Pro − , mutant of SNase with P11A, P31A, P42A, P47G, P56A, and P117G substitutions; SNase, wild-type staphylococcal nuclease with a sequence equivalent to that isolated from the Foggi strain of S. aureus).Article and publication are at

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Section: Discussionsupporting

confidence: 91%

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confidence: 99%

Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

et al. 2002

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“…Mutation analysis revealed that Val66, Ala69 and Ala90 in the β-barrel domain are fully native-like in the transition state. 41 In contrast, a Ca 2+ -binding site, which is located at the interface of theβ-barrel and the α-helical domains, is not yet formed at this state. 43 These findings indicate that the final steps of folding involve formation of specific docking interactions between the subdomains of SNase.…”

Section: Introductionmentioning

confidence: 93%

Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods

Maki¹,

Cheng²,

Долгих³

et al. 2007

Journal of Molecular Biology

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To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophans in an apolar environment. The kinetics of folding was observed by continuous-and stopped-flow fluorescence measurements over refolding times ranging from 100 μs to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophans in the β-barrel and the N-terminal part of the α-helical domain become partially shielded from the solvent at an early stage (< 1 ms), indicating that this region of the protein undergoes a rapid specific collapse and remains uncoupled from the rest of the α-helical domain until the late stages of folding. For several variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophans reach their buried native environment only during the late stages of folding. Other variants show more complex behavior with a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophans into close contact with intramolecular quenchers.

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“…Similar results have been obtained for a number of other proteins including α-lactalbumin [78] and apomyoglobin [79], giving rise to the proposal that the folding process proceeds via an intermediate with the principal characteristics of a "molten globule" (see Section 4.2). In a study of the refolding of native and mutant forms of staphylococcal nuclease, it was found that a transient kinetic intermediate formed within 10 msec [80]. This intermediate possessed only about 30% of the native ellipticity at 225 nm; in conjunction with hydrogen exchange experiments it was shown that this relatively low amplitude reflected formation of the 5-stranded β-sheet structural core of the N-terminal domain of the enzyme, with the helices (which contribute the bulk of the CD signal at 225 nm) formed at a later stage [81].…”

Section: The Nature Of Early Folding Intermediatesmentioning

confidence: 99%

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

Kelly

Price²

2000

CPPS

969

707

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Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as α-helix and β-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising "molten globule" intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.

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Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped‐flow circular dichroism

Cited by 26 publications

References 47 publications

Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

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