Early Events During Folding of Wild-type Staphylococcal Nuclease and a Single-tryptophan Variant Studied by Ultrarapid Mixing

2004 ◽  
Vol 338 (2) ◽  
pp. 383-400 ◽  
Author(s):  
Kosuke Maki ◽  
Hong Cheng ◽  
Dimitry A. Dolgikh ◽  
M.C.Ramachandra Shastry ◽  
Heinrich Roder
Keyword(s):  
Staphylococcal Nuclease ◽  
Wild Type

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

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?

scholarly journals Controlled Production of Stable Heterologous Proteins in Lactococcus lactis

2002 ◽  
Vol 68 (6) ◽  
pp. 3141-3146 ◽  
Author(s):  
A. Miyoshi ◽  
I. Poquet ◽  
V. Azevedo ◽  
J. Commissaire ◽  
L. Bermudez-Humaran ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Protein Production ◽  
Heterologous Protein ◽  
Nonstructural Protein ◽  
Inducible Promoter ◽  
Staphylococcal Nuclease ◽  
Poor Quality ◽  
Heterologous Proteins ◽  
Wild Type ◽  
Bovine Rotavirus

ABSTRACT The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P nisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.

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