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.


Abstract

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 degrees C (fluorescence) and 52.7+/-0.6 degrees 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 degrees and 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. Study holds ProTherm entries: 22504, 22505, 22506, 22507, 22508, 22509, 22510, 22511, 22512, 22513, 22514, 22515, 22516, 22517, 22518, 22519, 22520, 22521, 22522, 22523, 22524, 22525, 22526, 22527, 22528, 22529, 22530, 22531, 22532, 22533, 22534, 22535, 22536, 22537, 22538, 22539, 22540, 22541, 22542, 22543, 22544, 22545, 22546, 22547, 22548, 22549, 22550, 22551, 22552, 22553, 22554, 22555, 22556, 22557, 22558, 22559, 22560, 22561, 22562, 22563, 22564, 22565, 22566, 22567, 22568, 22569, 22570, 22571, 22572, 22573, 22574, 22575, 22576, 22577, 22578, 22579, 22580, 22581, 22582, 22583, 22584, 22585, 22586, 22587, 22588, 22589, 22590, 22591, 22592, 22593, 22594, 22595, 22596, 22597, 22598, 22599, 22600, 22601, 22602, 22603, 22604, 22605, 22606, 22607, 22608, 22609, 22610, 22611, 22612, 22613, 22614, 22615, 22616, 22617, 22618, 22619, 22620, 22621, 22622, 22623, 22624, 22625, 22626, 22627, 22628, 22629, 22630, 22631, 22632, 22633, 22634, 22635, 22636, 22637, 22638, 22639, 22640, 22641, 22642, 22643, 22644, 22645, 22646, 22647, 22648, 22649, 22650, 22651, 22652, 22653, 22654, 22655, 22656, 22657, 22658, 22659, 22660, 22661, 22662, 22663, 22664, 22665, 22666, 22667, 22668, 22669, 22670, 22671, 22672, 22673, 22674, 22675, 22676, 22677, 22678, 22679, 22680, 22681, 22682, 22683, 22684, 22685, 22686, 22687, 22688, 22689, 22690, 22691, 22692, 22693, 22694, 22695, 22696, 22697, 22698, 22699 Extra Details: Thermal unfolding; Residual structure; Melting temperature; van't Hoff enthalpy

Submission Details

ID: jmGztXvT3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:53 p.m.

Version: 1

Publication Details
Byrne MP;Stites WE,Biophys. Chem. (2007) 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. PMID:17134819
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 PDB Entries

Structure ID Release Date Resolution Structure Title
4WRD 2014-10-23T00:00:00+0000 1.65 Crystal structure of Staphylcoccal nulease variant Delta+PHS V66E L125E at cryogenic temperature
2LKV 2011-10-21T00:00:00+0000 0 Staphylococcal Nuclease PHS variant
2M00 2012-10-14T00:00:00+0000 0 Solution structure of staphylococcal nuclease E43S mutant in the presence of ssDNA and Cd2+
2OXP 2007-02-20T00:00:00+0000 2.0 Crystal Structure of Staphylococcal Nuclease mutant V66D/P117G/H124L/S128A
3D4W 2008-05-15T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant Delta+PHS A109R at cryogenic temperature
3D8G 2008-05-23T00:00:00+0000 1.99 Crystal structure of Staphylococcal nuclease variant Delta+PHS I72R at cryogenic temperature
3MVV 2010-05-04T00:00:00+0000 1.72 Crystal structure of Staphylococcal nuclease variant Delta+PHS F34A at cryogenic temperature
3QOJ 2011-02-10T00:00:00+0000 1.6 Cryogenic structure of Staphylococcal nuclease variant D+PHS/V23K
3QOL 2011-02-10T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant D+PHS/V23E at pH 6 determined at 100 K
3R3O 2011-03-16T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant Delta+PHS T62A at cryogenic temperature and with high redundancy

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