pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

Biochemistry ◽  
1990 ◽  
Vol 29 (10) ◽  
pp. 2564-2572 ◽  
Author(s):  
C. Nick Pace ◽  
Douglas V. Laurents ◽  
James A. Thomson
Keyword(s):  
Ph Dependence ◽  
Guanidine Hydrochloride ◽  
Ribonuclease A ◽  
Ribonuclease T1

Effect of peptide binding on amide proton exchange rates in the PDZ2 domain from human phosphatase hPTP1E

1998 ◽  
Vol 76 (2-3) ◽  
pp. 334-340 ◽  
Author(s):  
Irena Ekiel ◽  
Denis Banville ◽  
Shi Hsiang Shen ◽  
Kalle Gehring
Keyword(s):  
Exchange Rates ◽  
Ph Dependence ◽  
Guanidine Hydrochloride ◽  
Peptide Binding ◽  
Fluorescence Emission ◽  
Proton Exchange ◽  
Amide Proton ◽  
Amide Proton Exchange ◽  
C Terminus ◽  
Amide Exchange

Amide hydrogen-deuterium exchange rates were measured in the PDZ2 domain from human phosphatase hPTP1E by 1H-15N heteronuclear NMR spectroscopy. Protection factors were calculated for the slowly exchanging hydrogens in both the free PDZ2 domain and its complex with an octapeptide peptide, R-N-E-I-Q-S-L-V, derived from the C-terminus of the Fas receptor. Aside from a short α-helical region α1 (amino acids A-45 to D-49), the pattern of highly protected amides correlated well with the presence of hydrogen bonds in elements of the secondary structure. Hydrogen-bonded amides showed relatively fast exchange rates with half-lives of less than 9 h at pD 7.6 and 8°C. Protection factors, calculated as the ratio of theoretical (denatured) and observed exchange rates, showed less dispersion in maximal values than did the actual exchange rates. This behavior and the large pH dependence of the exchange rates suggest that amide exchange is close to the EX2 limit. In this limit, exchange of the most protected amides occurs through a global unfolding mechanism. The free energy of the unfolding calculated from the largest protection factors is 4.8 ± 0.4 kcal/mol (1 cal = 4.184 J). This ΔG° closely matches the value measured by experiments with guanidine hydrochloride and fluorescence emission spectroscopy. Peptide binding to PDZ2 resulted in mostly global effects and stabilized the folded domain by 1.4 kcal/mol.Key words: PDZ2 from hPTP1E, amide exchange, ligand binding, NMR.

Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A

Biochemistry ◽  
1987 ◽  
Vol 26 (26) ◽  
pp. 8620-8624 ◽  
Author(s):  
Satoshi Nishikawa ◽  
Hiroshi Morioka ◽  
Hyo J. Kim ◽  
Kayoko Fuchimura ◽  
Toshiki Tanaka ◽  
...  
Keyword(s):  
Ribonuclease A ◽  
Ribonuclease T1 ◽  
Histidine Residues

Denaturation of proteins. IV. N.M.R. studies of ribonuclease-A

1972 ◽  
Vol 25 (1) ◽  
pp. 209 ◽  
Author(s):  
JH Bradbury ◽  
NLR King
Keyword(s):  
Formic Acid ◽  
Deuterium Oxide ◽  
Guanidine Hydrochloride ◽  
Difference Spectrum ◽  
Potassium Thiocyanate ◽  
Ribonuclease A ◽  
Relative Increase ◽  
Intermediate Form ◽  
State Process ◽  
Major Transition

The denaturation of ribonuclease-A by the addition of urea, guanidine hydrochloride, formic acid, and potassium thiocyanate to solutions in water or D2O at 33� has been followed by nuclear magnetic resonance (N.M.R.) spectroscopy. ��The complex N.M.R. spectra at low field can be simplified greatly by a difference spectrum obtained by subtracting the spectrum obtained in deuterium oxide from the corresponding spectrum in water, whence the resonances of protons attached to nitrogen are isolated. Binding of urea and guanidine hydrochloride at concentrations well below that needed for unfolding is shown by modification of the C2 histidine resonances due to the histidines located at positions 12 and 119. This confirms that these denaturants inactivate the enzyme by binding at its active site as proposed by Barnard.The unfolding of ribonuclease by urea and guanidine hydrochloride at acid pH is shown to be a two-state process in which the fraction of unfolded molecules (cross-linked random coils) is calculated directly from the relative increase in heights of the various n.m.r, resonances. The unfolding in [D2]formic acid is characterized by the first (major) transition with a midpoint at 8% [D2] formic acid (v/v) and a second (minor) transition centred at 58% [D2]formic acid. In pure formic acid there is evidence of aggregate formation. An intermediate form characterized by a double methionine SCH3 resonance occurs during the first transition. There are therefore a minimum of five different states present during this unfolding. The major unfolding process produced by potassium thiocyanate is followed by a refolding to a non-native ordered form. This unfolding process is incomplete and three different

pH Dependence of the thermal unfolding of ribonuclease A

Biochemistry ◽  
1972 ◽  
Vol 11 (5) ◽  
pp. 879-883 ◽  
Author(s):  
Peter McPhie
Keyword(s):  
Ph Dependence ◽  
Ribonuclease A ◽  
Thermal Unfolding
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