Protein Science, Vol 3, Issue 11 1984-1991, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions

O. D. MONERA, C. M. KAY and R. S. HODGES
Department of Biochemistry and the Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl](1/2) values (average of {complex}3.5 M) and, as well, their {Delta}{Delta}G(u) values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea](1/2) values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the {Delta}{Delta}G(u) values correspondingly increased with the increasing differences in electrostatic interactions (20A - 15A5R, 1.5 kcal/mol; 20A - 10A10R, 3.7 kcal/mol; and 20A - 20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the uncharged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.
CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. A. Beckstead, K. Wong, V. Gupta, C.-P. L. Wan, V. R. Cook, R. B. Weinberg, P. M. M. Weers, and R. O. Ryan The C Terminus of Apolipoprotein A-V Modulates Lipid-binding Activity J. Biol. Chem., May 25, 2007; 282(21): 15484 - 15489. [Abstract] [Full Text] [PDF]

				M. Pruteanu, S. B. Neher, and T. A. Baker Ligand-Controlled Proteolysis of the Escherichia coli Transcriptional Regulator ZntR J. Bacteriol., April 15, 2007; 189(8): 3017 - 3025. [Abstract] [Full Text] [PDF]

				O. Garcia-Arribas, R. Mateo, M. M. Tomczak, P. L. Davies, and M. G. Mateu Thermodynamic stability of a cold-adapted protein, type III antifreeze protein, and energetic contribution of salt bridges Protein Sci., February 1, 2007; 16(2): 227 - 238. [Abstract] [Full Text] [PDF]

				C. D. Geierhaas, A. A. Nickson, K. Lindorff-Larsen, J. Clarke, and M. Vendruscolo BPPred: A Web-based computational tool for predicting biophysical parameters of proteins Protein Sci., January 1, 2007; 16(1): 125 - 134. [Abstract] [Full Text] [PDF]

				S. L. Flaugh, I. A. Mills, and J. King Glutamine Deamidation Destabilizes Human {gamma}D-Crystallin and Lowers the Kinetic Barrier to Unfolding J. Biol. Chem., October 13, 2006; 281(41): 30782 - 30793. [Abstract] [Full Text] [PDF]

				D. C. Vaz, J. R. Rodrigues, W. Sebald, C. M. Dobson, and R. M.M. Brito Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of Interleukin-4 Protein Sci., January 1, 2006; 15(1): 33 - 44. [Abstract] [Full Text] [PDF]

				A. Zarrine-Afsar, A. Mittermaier, L. E. Kay, and A. R. Davidson Protein stabilization by specific binding of guanidinium to a functional arginine-binding surface on an SH3 domain Protein Sci., January 1, 2006; 15(1): 162 - 170. [Abstract] [Full Text] [PDF]

				Y. Kumar, S. Muzammil, and S. Tayyab Influence of Fluoro, Chloro and Alkyl Alcohols on the Folding Pathway of Human Serum Albumin J. Biochem., October 1, 2005; 138(4): 335 - 341. [Abstract] [Full Text] [PDF]

				A. Sandberg, J. Leckner, and B. G. Karlsson Apo-azurin folds via an intermediate that resembles the molten-globule Protein Sci., October 22, 2004; 13(10): 2628 - 2638. [Abstract] [Full Text] [PDF]

				K. C. Engman, A. Sandberg, J. Leckner, and B. G. Karlsson Probing the influence on folding behavior of structurally conserved core residues in P. aeruginosa apo-azurin Protein Sci., October 22, 2004; 13(10): 2706 - 2715. [Abstract] [Full Text] [PDF]

				R. Kumar, N. P. Prabhu, M. Yadaiah, and A. K. Bhuyan Protein Stiffening and Entropic Stabilization in the Subdenaturing Limit of Guanidine Hydrochloride Biophys. J., October 1, 2004; 87(4): 2656 - 2662. [Abstract] [Full Text] [PDF]

				A. B. Cowley, M. Rivera, and D. R. Benson Stabilizing roles of residual structure in the empty heme binding pockets and unfolded states of microsomal and mitochondrial apocytochrome b5 Protein Sci., September 1, 2004; 13(9): 2316 - 2329. [Abstract] [Full Text] [PDF]

				M. Zakrzewska, D. Krowarsch, A. Wiedlocha, and J. Otlewski Design of fully active FGF-1 variants with increased stability Protein Eng. Des. Sel., August 1, 2004; 17(8): 603 - 611. [Abstract] [Full Text] [PDF]

				R. Perez-Jimenez, R. Godoy-Ruiz, B. Ibarra-Molero, and J. M. Sanchez-Ruiz The Efficiency of Different Salts to Screen Charge Interactions in Proteins: A Hofmeister Effect? Biophys. J., April 1, 2004; 86(4): 2414 - 2429. [Abstract] [Full Text] [PDF]

				S. M. Lu and R. S. Hodges Defining the minimum size of a hydrophobic cluster in two-stranded {alpha}-helical coiled-coils: Effects on protein stability Protein Sci., March 1, 2004; 13(3): 714 - 726. [Abstract] [Full Text] [PDF]

				M. G. Botelho, M. Gralle, C. L. P. Oliveira, I. Torriani, and S. T. Ferreira Folding and Stability of the Extracellular Domain of the Human Amyloid Precursor Protein J. Biol. Chem., September 5, 2003; 278(36): 34259 - 34267. [Abstract] [Full Text] [PDF]

				K. Prakash, S. Prajapati, A. Ahmad, S.K. Jain, and V. Bhakuni Unique oligomeric intermediates of bovine liver catalase Protein Sci., January 1, 2002; 11(1): 46 - 57. [Abstract] [Full Text] [PDF]

				S. Gianni, M. Brunori, and C. Travaglini-Allocatelli Refolding kinetics of cytochrome c551 reveals a mechanistic difference between urea and guanidine Protein Sci., August 1, 2001; 10(8): 1685 - 1688. [Abstract] [Full Text] [PDF]

				K. P. McGrath, M. M. Butler, C. M. DiGirolamo, D. L. Kaplan, W. A. Petka, and T. M. Laue Electrostatic Interactions in Leucine Zippers: Effects on Stability and Specificity of Interaction Journal of Bioactive and Compatible Polymers, July 1, 2000; 15(4): 334 - 356. [Abstract] [PDF]

				R. G. Eckenhoff and J. S. Johansson Molecular Interactions Between Inhaled Anesthetics and Proteins Pharmacol. Rev., December 1, 1997; 49(4): 343 - 368. [Abstract] [Full Text] [PDF]

				W. D. Kohn, O. D. Monera, C. M. Kay, and R. S. Hodges The Effects of Interhelical Electrostatic Repulsions between Glutamic Acid Residues in Controlling the Dimerization and Stability of Two-stranded alpha-Helical Coiled-coils J. Biol. Chem., October 27, 1995; 270(43): 25495 - 25506. [Abstract] [Full Text] [PDF]