Article pubs.acs.org/JPCB
Interactions of Urea with Native and Unfolded Proteins: A Volumetric Study Ikbae Son,† Yuen Lai Shek,† Anna Tikhomirova,† Eduardo Hidalgo Baltasar,‡ and Tigran V. Chalikian*,† †
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada ‡ Department of Physical Chemistry, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain S Supporting Information *
ABSTRACT: We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and αchymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea−protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea−protein interactions in both the native and unfolded states. We interpreted the ureadependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea−protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent−protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.
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INTRODUCTION Chemical denaturation has been a major component of biophysical research for over a century. Consequently, a great deal of effort has gone into understanding the mechanisms of modulation of the equilibrium between the native and unfolded protein species by stabilizing and destabilizing cosolvents.1−5 Although urea is the most common and frequently used cosolvent in protein studies, controversies exist about the molecular nature of its denaturing action. Currently, most researchers are leaning toward the direct as opposed to the indirect mechanism of urea-induced protein denaturation.5−8 The direct mechanism implies the existence of direct van der Waals or hydrogen bonding or other electrostatic interactions between urea and protein groups,9−11 while, in the indirect mechanism, urea exerts its influence via perturbation of the structure of water and the subsequent modification of protein− water interactions.12−14 Volumetric properties of solutes provide a wealth of thermodynamic information describing the entire spectrum of solute−solvent interactions.15−26 In particular, in a binary mixture consisting of the principal solvent and cosolvent, the volumetric properties of solutes reflect the differential solute− principal solvent and solute−cosolvent interactions.19,27,28 We have previously developed a statistical thermodynamics-based © XXXX American Chemical Society
algorithm that can be used in conjunction with experimental volumetric data to extract the thermodynamic parameters of elementary solute−cosolvent interactions.19,27,28 With this algorithm, we have conducted a systematic characterization of interactions of urea and glycine betaine with low-molecularweight model compounds and interactions of glycine betaine with native globular proteins.19,28−30 Our results collectively make argument in favor of extensive interactions of urea with all functional groups of proteins consistent with the direct mechanism of the effect of urea on protein stability.5,9−11,31−33 More recently, our theoretical investigation based on the volumetrically determined parameters for urea− and glycine betaine−protein interactions has revealed that the mode of action of a specific cosolvent is governed by an extremely subtle balance between the thermodynamic contributions of cavity formation and direct solute−cosolvent interactions.34 In this work, we expand this line of research to studying the solute−solvent interactions of four proteins, namely, apocytochrome c, hen egg white lysozyme, ribonuclease A, and α-chymotrypsinogen A, in Received: September 15, 2014 Revised: November 3, 2014
A
dx.doi.org/10.1021/jp509356k | J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
Table 1. Extinction Coefficients (M−1 cm−1) of the Proteins as a Function of Urea urea (M)
lysozyme (280 nm)
apocytochrome c (277 nm)
ribonuclease A (278 nm)
α-chymotrypsinogen A (280 nm)
0 1 2 3 4 5 6 7 8
37900 38200 38630 39060 39630 39900 40200 40480 40770
10800 10950 11040 11150 11270 11320 11420 11530 11650
9880 9850 9760 9220 8960 8860 8950 9000 9050
50630 50450 48270 46690 46860 46970 47290 48490 48220
measurements (
