Instrumentation for Automated Determination of Protein Stability

Stites, Wesley E.; Byrne, Michael; Aviv, J.; Kaplan, M.; Curtis, P.

doi:10.1006/abio.1995.1259

Cited by 31 publications

(29 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutagenesis and protein expression and purification were carried out as described elsewhere [29]. Each mutant, at protein concentration of approximately 50 g/ml in a buffer consisting of 25 mM phosphate, 100 mM NaCl, pH 7.0, was subjected to a thermal denaturation in our Aviv ATF-101 fluorometer (296 nm excitation, 325 nm emission), as previously described in detail [30,31]. Thermal denaturations using circular dichroism (CD) at 222 nm as a probe of structure were performed using a Jasco 700 spectropolarimeter equipped with a Peltier thermocontrol unit in the same buffer but at protein concentrations of approximately 80 g/ml in a standard 1 cm cuvette with stirring.…”

Section: Methodsmentioning

confidence: 99%

Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: Lack of evidence for other than a two state thermal denaturation

Byrne

Stites

2007

Biophysical Chemistry

View full text Add to dashboard Cite

It is unclear whether the thermal denaturation of staphylococcal nuclease is a two state, three state, or variable two state process. The thermal denaturation of wild-type staphylococcal nuclease was followed by tryptophan fluorescence and circular dichroism signal at 222 nm, forty-two and fourteen times respectively. Analysis of this data using a simple two state model gave melting temperatures of 53.0 ± 0.4°C (fluorescence) and 52.7 ± 0.6°C (CD) and van't Hoff enthalpies of 82.4 ± 2.6 kcal/ mol and 88.6 ± 4.2 kcal/mol. Ninety-seven mutants also had these parameters determined by both fluorescence and CD. The average difference between the melting temperatures was 1.05 ± 0.75°a nd the average difference between van't Hoff enthalpies was 1.6 ± 4.8 kcal/mol. These very similar results for the two spectroscopic probes of structure are discussed in the context of the different models that have been proposed for nuclease denaturation. It is concluded, for most nuclease variants, that the errors introduced by a two state assumption are negligible and either virtually all helical structure is lost in any initial unfolding event or any intermediate must have low stability.

show abstract

Section: Methodsmentioning

confidence: 99%

Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: Lack of evidence for other than a two state thermal denaturation

Byrne

Stites

2007

Biophysical Chemistry

View full text Add to dashboard Cite

show abstract

“…70 The protein sample was allowed to equilibrate for 20 minutes at each concentration of urea. Analysis of the data was done according to the linear extrapolation model as described: 45,46 DG ¼ DG8 2 m ½urea ð 5Þ…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Mechanism of Thermostabilization in a Designed Cold Shock Protein with Optimized Surface Electrostatic Interactions

Makhatadze

Loladze

Gribenko

et al. 2004

Journal of Molecular Biology

View full text Add to dashboard Cite

“…The data was treated as a two-state model for reversible denaturation in which each protein molecule exists as folded or unfolded, whereas intermediate states are only transiently populated. Data analysis was carried out as previously published (Shortle et al, 1990;Stites et al, 1995). This analysis yields three parameters: a protein's stability to reversible denaturation (AGHZ0), the rate of change of free energy with respect to GuHCl concentration (rnGuHCI or d(AG)/d [GuHCI]), and the concentration of guanidine hydrochloride at which half the protein molecules are denatured (C,,,).…”

Section: Fluorescence Solvent Denaturationsmentioning

confidence: 99%

“…An Aviv model ATF-IO1 fluorometer (Stites et al, 1995) equipped with a Peltier thermocontrol unit was used to follow the thermal unfolding of the BMH crosslinked dimers, as well as the A60C DBP-and K70C-mustard crosslinked dimers. Untreated and MCA-treated cysteine mutants were also thermally unfolded.…”

Section: Fluorescence Thermal Denaturationsmentioning

confidence: 99%

Chemically crosslinked protein dimers: Stability and denaturation effects

Byrne

Stites

1995

Protein Science

View full text Add to dashboard Cite

Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; I ,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethy1)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCI) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, e-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.

show abstract

Instrumentation for Automated Determination of Protein Stability

Cited by 31 publications

References 0 publications

Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: Lack of evidence for other than a two state thermal denaturation

Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: Lack of evidence for other than a two state thermal denaturation

Mechanism of Thermostabilization in a Designed Cold Shock Protein with Optimized Surface Electrostatic Interactions

Chemically crosslinked protein dimers: Stability and denaturation effects

Contact Info

Product

Resources

About