Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing.


A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations. Study holds ProTherm entries: 16975, 16976, 16977, 16978, 16979, 16980, 16981, 16982, 16983, 16984, 16985, 16986 Extra Details: Pseudo wild type, P47G, P117G, H124L. 0.5mM EDTA was added in the experiment. protein folding kinetics; stopped-flow; continuous-flow; fluorescence; ANS

Submission Details

ID: yssxxaf7

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Maki K;Cheng H;Dolgikh DA;Shastry MC;Roder H,J. Mol. Biol. (2004) Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing. PMID:15066439
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Thermonuclease P00644 NUC_STAAU
99.3 Thermonuclease Q5HHM4 NUC_STAAC
99.3 Thermonuclease Q6GIK1 NUC_STAAR
99.3 Thermonuclease Q6GB41 NUC_STAAS
99.3 Thermonuclease Q8NXI6 NUC_STAAW
99.1 Thermonuclease Q99VJ0 NUC_STAAM
99.1 Thermonuclease Q7A6P2 NUC_STAAN