Compressibility As a Means to Detect and Characterize Globular Protein States TIGRAN V

Proc. Natil. Acad. Sci. USA Vol. 93, pp. 1012-1014, February 1996 Biochemistry

Compressibility as a means to detect and characterize globular protein states TIGRAN V. CHALIKIAN AND KENNETH J. BRESLAUER* Department of Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-1)939 Comnninicated by SteplVen J. Benkovic, Pennsylvania State University, University Park, PA, October 23, 1995

ABSTRACT We report compressibility data on single- inaccessible core and its coefficient of adiabatic compress- domain, globular proteins which suggest a general relation- ibility, OM (7): ship between protein conformational transitions and Ak', the change in the partial specific adiabatic compressibility kM = 13MVM/M, [2] which accompanies the transition. Specifically, we find transitions between native and compact intermediate states where M is the molecular weight of the protein. to be accompanied by small increases in ks of + (1-4) x 10-6 The hydration contribution, kh, reflects the decrease in the cm3 g-'bar-l (1 bar = 100 kPa). By contrast, transitions compressibility of the solvent which results from interactions between native and partially unfolded states are accompa- between the surface atomic groups of the protein and the nied by small decreases in ks of -(3-7) x 10-6 cm3g-'bar-', surrounding water molecules and can be calculated from the while native-to-fully unfolded transitions result in large de- expression (7, 8): creases in k' of -(18-20) x 10-6 cm3.g-'-bar-'. Thus, for the single-domain, globular proteins studied here, changes in ks kh = m - SAj&j, [31 correlate with the type of transition being monitored, independent of the specific protein. Consequently, k' measure- where SA, is the solvent-accessible surface area of the ith ments may provide a convenient approach for detecting the residue and Ksi is the compressibility contribution of 1 A2 of existence of and for defining the nature of protein transitions, this surface of the ith residue. In general, for globular proteins, while also characterizing the hydration properties of individ- the sum -i SA,Ksi yields negative values for the hydration ual protein states. contribution, kh, to the partial compressibility, although the sign and magnitude of Ksi for an individual residue depend on Depending on solution conditions, globular proteins can as- the experimental conditions and the type of the residue (8). sume a range of conformational states, including native (N), Based on these definitions and the relationship expressed by compact intermediate (CI), partially unfolded (PU), and fully Eq. 1, two generalizations emerge. First, the more rigid the unfolded (FU) (1-5). The N state of a globular protein is interior of a globular protein (in other words, the tighter its characterized by a unique tertiary structure, with tightly internal packing), the smaller its intrinsic compressibility, kM, packed amino acid residues buried within a solvent- and, consequently, the smaller the total partial specific adia- inaccessible protein interior. By contrast, the CI state, which batic compressibility, k°, of the protein. Second, the more includes molten globules (MGs), is characterized by a lack of protein atomic groups that are exposed to the solvent (in other rigid tertiary structure, a high content of secondary structure, words, the higher the total solvent-accessible surface area), the and a sizable core of nonpolar groups which are packed less more negative the hydration contribution, kh, and, conse- tightly than in the N state (4, 5). The PU state, which we define quently, the smaller the total value of k'. Given these inter- as the ensemble of partially unfolded conformations that can relationships, we present below the results of ks measurements be detected between the CI and FU states, is characterized by we have conducted on a range of globular proteins, beginning a lack of both tertiary and secondary structural elements, with with cytochrome c. a highly fluctuating and very loosely packed hydrophobic core. For cytochrome c, we find the partial specific adiabatic The FU state is random coil-like, with all atomic groups being compressibility, k', of the protein to vary significantly with the solvent accessible (2). nature of the protein conformational state present in solution Clearly, it would be very useful to identify an experimental (9). The following rank order emerges: k' (CI) > ks (N) > k' observable that could both detect and uniquely characterize (PU) > k' (FU). To be specific, for cytochrome c the acid- protein states. Based on the results reported here, we believe induced N-to-Cl transition is accompanied by a small positive that the partial specific adiabatic compressibility, k', of a change in ks of + 1.7 x 10-6 cm3g-'-bar-', and the acid- protein fulfills these requirements. Recall that the k' of a induced N-to-PU transition is accompanied by a small negative solute is a linear function of the isothermal pressure derivative change in k' of -3.9 x 10-6 cm3g- '-bar-l. We calculate the of its partial specific volume, v° (6). For globular proteins, the N-to-FU transition to be accompanied by a large negative value of k' is the sum of a positive intrinsic contribution, kM, change in k' of -20 x 10-6 cm3 g-l bar-1. These Ak' values and a negative hydration contribution, kh (7, 8): can be rationalized in terms of changes in a positively con- tributing intrinsic component, kM, and a negatively contribut- ko kM + kh- [1] ing hydration component, kh, with the latter diminishing with increasing protein hydration (ref. 8 and references therein; ref. The intrinsic contribution, kM, reflects the imperfect 9). For cytochrome c, we find the following trend in kh as a packing of the polypeptide chain(s) within the solvent- function of protein state: kh(N) > kh(CI) > kh(PU) > kh(FU). inaccessible interior of proteins. As shown in Eq. 2 below, the The positive contribution of kM increases as the interior value of kM is related to the volume, VM, of the water- packing of a protein decreases. For cytochrome c, we find that

The publication costs of this article were defrayed in part by page charge Abbreviations: N, native; CI, compact intermediate; PU, partially payment. This article must therefore be hereby marked 'adv'ertisenemnt" in unfolded; FU, fully unfolded; MG, molten globule. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1012 Downloaded by guest on September 23, 2021 Biochemistry: Chalikian and Breslauer Proc. Natl. Acad. Sci. USA 93 (1996) 1013 kM increases for the N-to-Cl and N-to-PU transitions but is buried residues of the N state become exposed to the solvent reduced almost to zero for the N-to-FU transition (9). In short, in the CI state (4). Consequently, the N-to-Cl transition our data on cytochrome c reveal that the value of k' and its should be accompanied by an increase in the accessible components, kM and kh, depend on the predominant protein surface area, SA, thereby resulting in a decrease in the state present in solution. hydration contribution, kh. Consistent with this expectation, To assess the generality, if any, of a relationship between the we find the acid-induced N-to-MG transition of cytochrome sign and magnitude of k' and the conformational state of a c to be accompanied by an -2-fold decrease in kh (9)- protein, we also have measured changes in compressibility that Furthermore, although the volume of the water-inaccessible accompany conformational transitions in three other small, core, VM, also should decrease upon a N-to-Cl transition, the single-domain, globular protein systems. The resulting data, intrinsic coefficient of adiabatic compressibility, ,BM, of the including those for cytochrome c, are listed in Table 1, along core should increase due to loosening of the interior packing. with the few k' values that can be found in the literature. The Consistent with this expectation, we find the acid-induced following features should be noted. All four of the N-to-Cl N-to-MG transition of cytochrome c to be accompanied by (e.g., MG) transitions are accompanied by small increases in a 4.5-fold increase in ,BM (9). As we have shown for cyto- ko of +(1-4) x 10-6 cm3g-'-bar-'. All three of the N-to-PU chrome c (9), this increase in ,BM prevails over the decrease transitions are accompanied by small decreases in ko of - (3-7) in VM, thereby resulting in a net increase in the intrinsic I x 10-6 cm3 g- bar- The N-to-FU transition of ribonuclease contribution, kM, to the partial compressibility (see Eq. 2). A is accompanied by a large decrease in k' of -18 x 10-6 For the N-to-MG transitions examined here, this increase in cm3.g- lbar- 1, which is in excellent agreement with our cal- kM is higher in absolute value than the decrease in kh. culated value of -20 x 10-6 cm3 g-l bar- for the N-to-FU Consequently, consistent with Eq. 1, we observe a net transition of cytochrome c (9). increase in k' of (1-4) x 10-6 cm3 g- Ibar-i for the N-to-CI In the aggregate, these results suggest a general relationship transitions of the globular proteins we have examined (see between the sign and magnitude of Ako and the protein state. Table 1). As discussed below, the origins of such a general relationship When the N state of a globular protein is converted into a can be understood in terms of the properties of the various PU state (the N-to-PU transition), a substantial increase in SA states that globular proteins can assume. should occur, with about 70% of the surface area expected for The N state of a globular protein is the most compact a fully extended chain becoming exposed to the solvent (9, 19). conformation amongst the possible thermodynamic states (N, This exposure should cause a large decrease in kh, although a CI, PU, and FU). This feature causes the N state to exhibit the water-inaccessible core, probably mostly hydrophobic, still is lowest solvent-accessible surface area, SA, and, consequently, preserved. The intrinsic volume, VM, of this preserved core in the highest (least negative) hydration contribution, kh, to the the PU state, however, should be small, while the coefficient partial compressibility. The native state also exhibits the of adiabatic compressibility, 13M, of the core should be high due largest volume for the water-inaccessible core, VM, as well as to it being loosely packed. Consequently, although the total the most tightly packed core, with the coefficient of adiabatic value of the intrinsic contribution, kM, may increase upon the compressibility, g3M, of this core being low and close to that of N-to-PU transition [as we find for the acid induced N-to-PU organic solids (7). transition of cytochrome c (9)], the absolute value of the When this N state of a globular protein is converted into decrease in kh due to solvent exposure prevails over any a CI state such as a MG (the N-to-CT transition), some of the increase in kM, since we observe moderate net decreases in ks Table 1. Partial specific adiabatic compressibilities, k', of globular proteins and the changes in compressibility, Aks, which accompany conformational transitions at 25°C Value x 106, cm3-g- l bar- Transition ko Aks Ref. N-to-MG of cytochrome Ca; acid-induced at 25°C in 200 mM CsCl 2.5 1.7 9 N-to-MG of a-lactalbumin6; acid-induced at 25°C in 2 mM CaC12 2.7 1.1 * N-to-Cl of a-chymotrypsinogen Ac; temperature-induced at pH 2.0 in 10 mM NaCl 3.2 3.9t * 2.6t 10 N-to-Cl of ribonuclease Ad; temperature induced at pH 1.9 0.9 4.Ot 11 N-to-PU of cytochrome Cc; acid-induced at 250C 2.5 -3.9 9 N-to-PU of cytochrome Cf; base-induced at 25°C 2.5 -3.8 22 N-to-PU of myoglobing; acid-induced at 25°C in 10 mM KCl 7.1 -6.8 12 -4.8 13 N-to-FU of ribonuclease Ah; Gdn HCl-induced at 25°C in 0.2 M CsCl 0.9 -18 11 aCharacterized as a N-to-MG transition by Ohgushi and Wada (14). bCharacterized as a N-to-MG transition by Dolgikh et al. (15). cCharacterized as a N-to-CT transition based on CD measurements (*) at acidic pH, which reveal that heat-denatured a-chymotrypsinogen A lacks tertiary structure while retaining secondary structure. '-Characterized as a N-to-CT transition based on CD spectra, small-angle x-ray scattering, and Fourier transform infrared spectroscopy (16, 17), which reveal that thermally denatured ribonuclease A has compact dimensions and retains residual secondary structure. cCharacterized as a N-to-PU transition based on CD measurements (9, 18), which reveal that, at low salt concentrations, the acid-denatured state of cytochrome c lacks both secondary and tertiary structure. tCharacterized as a N-to-PU transition based on CD measurements (22), which reveal that, at low salt concentrations, the base-denatured state of cytochrome c lacks both secondary and tertiary structure. gCharacterized as a N-to-PU transition based on CD measurements (*), which reveal that, at low salt concentrations, the acid-denatured state of myoglobin lacks both secondary and tertiary structure. hCharacterized as a N-to-FU transition based on the coincidence between the reported (12) and calculated (9) values of Aks. *T.V.C., V. S. Gindikin, D. Anafi, and K.J.B., unpublished data. tExtrapolated to 25°C. Downloaded by guest on September 23, 2021 1014 Biochemistry: Chalikian and Breslauer Proc. Natl. Acad. Sci. USA 93 (1996) of (3-7) x 10-6 cm3.g-lbar- for the N-to-PU transitions 5. Kim, P. S. & Baldwin, R. L. (1990) Annu. Rev. Biochem. 59, listed in Table 1 (see Eq. 1). 631-660. When the N state of a globular protein is converted into a 6. Hoiland, H. (1986) in Thermodynamic Data for Biochemistry and FU state (the N-to-FU transition), complete solvent exposure Biotechnology, ed. Hinz, H.-J. (Springer, Berlin), pp. 129-147. of the formally buried residues occurs. In the absence of a 7. Kharakoz, D. P. & Sarvazyan, A. P. (1993) Biopolymers 33, 11- solvent-inaccessible core, k' of the FU state should be deter- 26. 8. Chalikian, T. V., Sarvazyan, A. P. & Breslauer, K. J. (1994) Bio- mined exclusively by a large negative kh term. Consistent with phys. Chem. 51, 89-109. this expectation, we find the N-to-FU transition to be accom- 9. Chalikian, T. V., Gindikin, V. S. & Breslauer, K. J. (1995).J. Mol. panied by a large decrease in k' of about 20 x 10-6 Biol. 250, 291-306. cm3.g- 'bar- l 10. Kharakoz, D. P. & Sarvazyan, A. P. (1980) Stud. Biophys. 79, As noted above, our results suggest a general relationship 179-180. between the sign and magnitude of Ak' and the protein state. 11. Tamura, Y. & Gekko K. (1995) Biochemistry 34, 1878-1884. We propose that this relationship can be understood in terms 12. Kharakoz, D. P. & Karshikov, A. (1984) in Proceedings of the of the foregoing discussion. Significantly, however, the exis- International Symposium on theAcoustical Properties ofBiological tence of the empirical relationship and its utility do not depend Objects, ed. Sarvazyan, A. P. (Inst. of Biol. Phys., Acad. Sci. on the details of our interpretation. U.S.S.R. Press, Pushchino, U.S.S.R.), pp. 17-20. In summary, we find that changes in k' for single-domain, 13. Leung, W. P., Cho, K. C., Lo, Y. M. & Choy, C. L. (1986) Bio- globular proteins appear to correlate with the type of transi- chim. Biophys. Acta 870, 148-153. tion being monitored, independent of the specific globular 14. Ohgushi, M. & Wada, A. (1983) FEBS Lett. 164, 21-42. to 15. Dolgikh, D. A., Abaturov, L. V., Bolotina, I. A., Brazhnikov, protein under study. This property of Ak' may prove be a E. V., Bychkova, V. E., Gilmanshin, R. I., Lebedev, Y. O., Semi- common thermodynamic feature of small globular proteins, sotnov, G. V., Tiktopulo, E. I. & Ptitsyn, 0. B. (1985) Eur. Bio- similar to the convergence of the changes in enthalpy (at phys. J. 13, 109-121. 110°C) and entropy (at 112°C) which accompany heat dena- 16. Sosnick, T. R. & Trewhella, J. (1992) Biochemistry 31, 8329- turation of such proteins (19-21). Consequently, ks measure- 8335. ments may provide a convenient approach for detecting the 17. 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