Chemically crosslinked protein dimers: stability and denaturation effects.


Abstract

Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; 1,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethyl)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCl) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, epsilon-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins. Study holds ProTherm entries: 9199, 9200, 9201, 9202, 9203, 9204, 9205, 9206, 9207, 9208, 9209, 9210, 9211, 9212, 9213, 9214, 9215, 9216, 9217, 9218, 9219, 9220, 9221, 9222, 9223, 9224, 9225, 9226, 9227, 9228, 9229, 9230, 9231, 9232, 9233, 9234 Extra Details:

Submission Details

ID: RGmcUrpd3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Byrne MP;Stites WE,Protein Sci. (1995) Chemically crosslinked protein dimers: stability and denaturation effects. PMID:8580845
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
99.1 Thermonuclease Q99VJ0 NUC_STAAM
99.1 Thermonuclease Q7A6P2 NUC_STAAN
99.3 Thermonuclease Q6GB41 NUC_STAAS
99.3 Thermonuclease Q8NXI6 NUC_STAAW
99.3 Thermonuclease Q6GIK1 NUC_STAAR