Does proline isomerization shape the folding funnel of the wild type and mutant staphylococcal nuclease?

1999 ◽  
Author(s):  
Tian Yow Tsong ◽  
Zheng-Ding Su
Keyword(s):  
Staphylococcal Nuclease ◽  
Wild Type ◽  
Proline Isomerization

NMR and Mutagenesis Investigations of a Model Cis: Trans Peptide tsomerization Reaction: Xaa116-Pro117of Staphylococcal Nuclease and its Role in Protein Stability and Folding

1997 ◽  
Author(s):  
A.P. Hinck ◽  
W.F. Walkenhorst
Keyword(s):  
Protein Stability ◽  
Time Scale ◽  
Chemical Exchange ◽  
Chemical Shifts ◽  
Peptide Bond ◽  
Staphylococcal Nuclease ◽  
Isomerization Reaction ◽  
Molecular Heterogeneity ◽  
Proline Isomerization ◽  
Imino Acids

The slow rates of peptide bond isomerization in imino acids and the substantial population of the cis peptide bond isomer in Xaa-Pro linkages in peptides were first recognized in NMR studies of proline-containing model compounds (Maia et al., 1971). The important role of this isomerization in protein stability and folding (reviewed by Kim and Baldwin, 1982, 1990; Schmid, 1993) were recognized several years later (Brandts et al., 1975) and the biological relevance of this process was substantiated by the discovery of a ubiquitous enzyme that catalyzes Xaa-Pro peptide bond isomerization (Fischer et al., 1984, 1989; Takahashi et al., 1989). The strict evolutionary conservation of some prolyl residues and the observation that the kinetics of interconversion between alternative functional forms of some systems is consistent with the time scale of proline isomerization suggest that proline isomerization may play a wide role in protein structure and function. Suggestive examples include the sodium pump of Escherichia coli, the disulfide isomerase/thioredoxin class of enzymes, concanavalin A, and bovine prothrornbin fragment I (Brown et al., 1977; Marsh et al, 1979; Dunker, 1982; Brandland Deber, 1986; Langsetmo et al, 1989). NMR spectroscopy is one of the most suitable tools for studying this isomerization reaction. The rates generally are slow on the time scale of NMR chemical shifts but, in favorable cases, are comparable to longitudinal relaxation rates so that the isomerization process can be investigated by chemical exchange spectroscopy. NMR data obtained on calbindin D9k (Chazin et al., 1989), insulin (Higgins et al., 1988), and staphylococcal nuclease (nuclease) as discussed below have shown that each exists in solution under native conditions as a mixture of slowly exchanging conformers. The fact that dynamic molecular heterogeneity in nuclease was first observed in the laboratory of Oleg Jardetzky, as manifested by splitting of the histidyl 1H ε1 resonance from His46 in one-dimensional 1H NMR spectra recorded at 100 MHz (Markley et al., 1970), makes this topic particularly appropriate to a volume celebrating his scientific contributions.

Pressure-jump Relaxation Kinetics of Unfolding and Refolding Transitions of Staphylococcal Nuclease and Proline Isomerization Mutants

1996 ◽  
Author(s):  
Gedlminas J. A. Vidugiris ◽  
Raj Thomas
Keyword(s):  
Globular Proteins ◽  
Staphylococcal Nuclease ◽  
Site Directed Mutagenesis ◽  
Pressure Jump ◽  
Wild Type ◽  
Relaxation Kinetics ◽  
Signal Activity ◽  
In The Wild ◽  
Pressure Jump Relaxation ◽  
Kinetics Of

We present here the first report of the pressure dependence of pressure-jump relaxation kinetics for protein folding transitions. We have studied the relaxation kinetics for the unfolding/refolding of wild-type Staphylococcal nuclease and have found that the relaxation kinetics observed at high pressure are much slower than those observed by pH or denaturant jumps at atmospheric pressure. This indicates that these processes have large, positive values for the activation volumes, most likely stemming from exclusion of solvent from a transition state that is less well packed than the native state. We examined the pressure-jump relaxation kinetics of three single-site mutations in nuclease that lead to alterations in the interactions between the two domains of the protein and changes in the equilibrium constant for isomerization of the lysine-116 to proline 117 peptide bond away from the cis form that predominates in the wild-type enzyme. At comparable pressures, the relaxation times for these mutants were significantly shorter than those observed for the wild type, indicating lower values of the activation volumes. We propose that these mutations cause a decrease in the cooperativity of the unfolding of the two domains, leading to a decrease in the degree of solvent exclusion at the rate-limiting step. The mechanism by which a particular amino acid sequence determines the fold and stability of globular proteins remains one of the most interesting and important unresolved issues in biophysical chemistry. The approaches to increasing our understanding of this phenomenon typically have involved perturbation of the proteins by chemical means or by temperature extremes. The equilibrium or time-dependent responses to these perturbations are then monitored (using a spectroscopic signal, activity, or some other observable) to extract the energetic or kinetic aspects of the unfolding or refolding transitions. Another means of perturbing the system is to modify the protein itself, either chemically or by site-directed mutagenesis, and to assess the effects of modification on the equilibrium or kinetic folding or refolding profiles. This approach has generated a great deal of information about small globular proteins that denature reversibly.

scholarly journals 1P071 Glycerol effects for the folding reaction of wild-type Staphylococcal nuclease

2005 ◽  
Vol 45 (supplement) ◽  
pp. S49
Author(s):  
T. Baba ◽  
H. Kamikubo ◽  
M. Onitsuka ◽  
Y. Yamazaki ◽  
Y. Imamoto ◽  
...  
Keyword(s):  
Staphylococcal Nuclease ◽  
Wild Type

scholarly journals Kinetic and thermodynamic consequences of the removal of theCys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase

1997 ◽  
Vol 321 (2) ◽  
pp. 413-417 ◽  
Author(s):  
Marc VANHOVE ◽  
Gilliane GUILLAUME ◽  
Philippe LEDENT ◽  
John H. RICHARDS ◽  
Roger H. PAIN ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Guanidine Hydrochloride ◽  
Tryptophan Fluorescence ◽  
Disulphide Bond ◽  
Mutant Protein ◽  
Wild Type ◽  
High Concentration ◽  
Proline Isomerization ◽  
Drastic Increase ◽  
Time Courses

Class A α-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 α-lactamase, this bond was removed by introducing a Cys-77 → Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N ↔ H ↔ U). Both the folded mutant protein (N) and, to a lesser extent, the thermodynamically stable intermediate, H, were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.

Temperature- and Pressure-Induced Unfolding of a Mutant of Staphylococcal Nuclease A

1996 ◽  
Author(s):  
Maurice R. Eftink ◽  
Glen D. Ramsay
Keyword(s):  
Free Energy ◽  
Thermodynamic Stability ◽  
Structural Features ◽  
Staphylococcal Nuclease ◽  
Tryptophan Residue ◽  
Hybrid Protein ◽  
Wild Type ◽  
Free Energy Surface ◽  
Native State ◽  
Maximum Stability

Nuclease conA is a hybrid version of Staphylococcal nuclease that contains a six amino acid (β-turn substitute from concanavalin A. This hybrid protein has a much lower thermodynamic stability than does the wild-type protein. This enables the unfolding of the protein to be achieved easily by several types of perturbations. From temperature-, pressure-, and denaturant-induced unfolding studies, we have found the free energy change for unfolding, ΔG°un, to be approximately 1.4 kcal mo−1 at pH 7, 0.1 M NaCI, and 20 °C, as compared to a thermodynamic stability of approximately 5.5–6 kcal mo−1 for wild-type nuclease A. Due to its reduced thermodynamic stability, nuclease conA also shows evidence of unfolding at low-temperature (cold denaturation), with a temperature of maximum stability of 13–15 °C. The thermal unfolding of nuclease conA is shown to be two-state by simultaneous measurement of fluorescence and CD changes as a function of temperature, using a modified AVIV CD instrument. Increased hydrostatic pressure unfolds nuclease conA in what appears to be a two-state manner, with an apparent of ΔV°un approximately —100 ml mol−1. From studies of the pressure (p)-induced unfolding of this hybrid protein as a function of temperature (T), we can define the complete p-T free energy surface for the unfolding transition. In auxiliary studies, we have characterized the fluorescence intensity decay and anisotropy decay of the single tryptophan residue (Trp-140) of nuclease conA in the native state and in the unfolded state induced by temperature, pressure, and denaturant. For each type of perturbation, there is a red shift in fluorescence, a lowering of the mean fluorescence lifetime, and a lowering of the rotational correlation time of the tryptophan residue to a value of ~1 ns (compared to 10–15 ns for the native state). The thermodynamics of the unfolding of proteins has received renewed interest in recent years, owing to the availability of a rich variety of mutant proteins and to advances in our understanding of their structural features. Among the questions being asked are, What are the relative energetic contributions of the hydrophobic effect and other interaction forces?

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