| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843, USA
2 Engelhardt Institute of Molecular Biology, Moscow 119991, Russia
3 Department of Biochemistry and Biophysics and The Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843, USA
Reprint requests to: C. Nick Pace, Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843–1114, USA; e-mail: nickpace{at}tamu.edu; fax: 979-847-9481.
The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.
Keywords: Ribonuclease Sa; isoelectric pH; net charge; electrostatic interactions; protein solubility; enzyme activity; protein stability
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Tjong and H.-X. Zhou Prediction of Protein Solubility from Calculation of Transfer Free Energy Biophys. J., September 15, 2008; 95(6): 2601 - 2609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Thurlkill, G. R. Grimsley, J. M. Scholtz, and C. N. Pace pK values of the ionizable groups of proteins Protein Sci., May 1, 2006; 15(5): 1214 - 1218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Trefethen, C. N. Pace, J. M. Scholtz, and D. N. Brems Charge-charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa Protein Sci., July 1, 2005; 14(7): 1934 - 1938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Fuchs and R. T. Raines Polyarginine as a multifunctional fusion tag Protein Sci., June 1, 2005; 14(6): 1538 - 1544. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. M. Chow, A. M. Ellisdon, L. D. Cabrita, and S. P. Bottomley Polyglutamine Expansion in Ataxin-3 Does Not Affect Protein Stability: IMPLICATIONS FOR MISFOLDING AND DISEASE J. Biol. Chem., November 12, 2004; 279(46): 47643 - 47651. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. K. Mosavi and Z.-y. Peng Structure-based substitutions for increased solubility of a designed protein Protein Eng. Des. Sel., October 1, 2003; 16(10): 739 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. I. Yakovlev, V. A. Mitkevich, K. L. Shaw, S. Trevino, S. Newsom, C. N. Pace, and A. A. Makarov Contribution of active site residues to the activity and thermal stability of ribonuclease Sa Protein Sci., October 1, 2003; 12(10): 2367 - 2373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Schmittschmitt and J. M. Scholtz The role of protein stability, solubility, and net charge in amyloid fibril formation Protein Sci., October 1, 2003; 12(10): 2374 - 2378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Takano, J. M. Scholtz, J. C. Sacchettini, and C. N. Pace The Contribution of Polar Group Burial to Protein Stability Is Strongly Context-dependent J. Biol. Chem., August 22, 2003; 278(34): 31790 - 31795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-B. Wong, C.-F. Lee, S.-H. Chan, T.-Y. Leung, Y. W. Chen, and M. Bycroft Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer Protein Sci., July 1, 2003; 12(7): 1483 - 1495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Tollinger, K. A. Crowhurst, L. E. Kay, and J. D. Forman-Kay Site-specific contributions to the pH dependence of protein stability PNAS, April 15, 2003; 100(8): 4545 - 4550. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mozo-Villarias, J. Cedano, and E. Querol A simple electrostatic criterion for predicting the thermal stability of proteins Protein Eng. Des. Sel., April 1, 2003; 16(4): 279 - 286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Sevcik, L. Urbanikova, P. A. Leland, and R. T. Raines X-ray Structure of Two Crystalline Forms of a Streptomycete Ribonuclease with Cytotoxic Activity J. Biol. Chem., November 27, 2002; 277(49): 47325 - 47330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. N. Ilinskaya, F. Dreyer, V. A. Mitkevich, K. L. Shaw, C. N. Pace, and A. A. Makarov Changing the net charge from negative to positive makes ribonuclease Sa cytotoxic Protein Sci., October 1, 2002; 11(10): 2522 - 2525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Dong and H.-X. Zhou Electrostatic Contributions to T4 Lysozyme Stability: Solvent-Exposed Charges versus Semi-Buried Salt Bridges Biophys. J., September 1, 2002; 83(3): 1341 - 1347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. K. Lee, C. A. Fitch, and B. Garcia-Moreno E. Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein Protein Sci., May 1, 2002; 11(5): 1004 - 1016. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Maldonado, M. P. Irun, L. A. Campos, J. A. Rubio, A. Luquita, A. Lostao, R. Wang, B. Garcia-Moreno E., and J. Sancho Salt-induced stabilization of apoflavodoxin at neutral pH is mediated through cation-specific effects Protein Sci., May 1, 2002; 11(5): 1260 - 1273. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
