Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering.

Protein Sci. 1997 February; 6(2): 422–430.

PMCID: PMC2143659

M. Kataoka, K. Kuwajima, F. Tokunaga, and Y. Goto

Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Japan.

M. Kataoka: kataoka/at/ess.sci.osaka-u.ac.jp

This article has been cited by other articles in PMC.

Abstract

A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the alpha-lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of alpha-lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of alpha-lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 A for the native state to 60 A for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the alpha-lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the alpha-lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.

Full Text

The Full Text of this article is available as a PDF (1.9M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

Acharya, KR; Stuart, DI; Walker, NP; Lewis, M; Phillips, DC. Refined structure of baboon alpha-lactalbumin at 1.7 A resolution. Comparison with C-type lysozyme. J Mol Biol. 1989 Jul 5;208(1):99–127. [PubMed]
Alexandrescu, AT; Evans, PA; Pitkeathly, M; Baum, J; Dobson, CM. Structure and dynamics of the acid-denatured molten globule state of alpha-lactalbumin: a two-dimensional NMR study. Biochemistry. 1993 Feb 23;32(7):1707–1718. [PubMed]
Carra, JH; Anderson, EA; Privalov, PL. Three-state thermodynamic analysis of the denaturation of staphylococcal nuclease mutants. Biochemistry. 1994 Sep 6;33(35):10842–10850. [PubMed]
Chyan, CL; Wormald, C; Dobson, CM; Evans, PA; Baum, J. Structure and stability of the molten globule state of guinea-pig alpha-lactalbumin: a hydrogen exchange study. Biochemistry. 1993 Jun 1;32(21):5681–5691. [PubMed]
Creighton, TE; Darby, NJ; Kemmink, J. The roles of partly folded intermediates in protein folding. FASEB J. 1996 Jan;10(1):110–118. [PubMed]
Damaschun, G; Damaschun, H; Gast, K; Gernat, C; Zirwer, D. Acid denatured apo-cytochrome c is a random coil: evidence from small-angle X-ray scattering and dynamic light scattering. Biochim Biophys Acta. 1991 Jun 24;1078(2):289–295. [PubMed]
Damaschun, G; Damaschun, H; Gast, K; Misselwitz, R; Müller, JJ; Pfeil, W; Zirwer, D. Cold denaturation-induced conformational changes in phosphoglycerate kinase from yeast. Biochemistry. 1993 Aug 3;32(30):7739–7746. [PubMed]
Dill, KA; Shortle, D. Denatured states of proteins. Annu Rev Biochem. 1991;60:795–825. [PubMed]
Dolgikh, DA; Abaturov, LV; Bolotina, IA; Brazhnikov, EV; Bychkova, VE; Gilmanshin, RI; Lebedev YuO; Semisotnov, GV; Tiktopulo, EI; Ptitsyn, OB, et al. Compact state of a protein molecule with pronounced small-scale mobility: bovine alpha-lactalbumin. Eur Biophys J. 1985;13(2):109–121. [PubMed]
Dolgikh, DA; Gilmanshin, RI; Brazhnikov, EV; Bychkova, VE; Semisotnov, GV; Venyaminov SYu; Ptitsyn, OB. Alpha-Lactalbumin: compact state with fluctuating tertiary structure? FEBS Lett. 1981 Dec 28;136(2):311–315. [PubMed]
Doniach, S; Bascle, J; Garel, T; Orland, H. Partially folded states of proteins: characterization by X-ray scattering. J Mol Biol. 1995 Dec 15;254(5):960–967. [PubMed]
Ewbank, JJ; Creighton, TE. The molten globule protein conformation probed by disulphide bonds. Nature. 1991 Apr 11;350(6318):518–520. [PubMed]
Fersht, AR. Optimization of rates of protein folding: the nucleation-condensation mechanism and its implications. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10869–10873. [PubMed]
Fink, AL. Compact intermediate states in protein folding. Annu Rev Biophys Biomol Struct. 1995;24:495–522. [PubMed]
Flanagan, JM; Kataoka, M; Fujisawa, T; Engelman, DM. Mutations can cause large changes in the conformation of a denatured protein. Biochemistry. 1993 Oct 5;32(39):10359–10370. [PubMed]
Flanagan, JM; Kataoka, M; Shortle, D; Engelman, DM. Truncated staphylococcal nuclease is compact but disordered. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):748–752. [PubMed]
Gast, K; Damaschun, H; Misselwitz, R; Müller-Frohne, M; Zirwer, D; Damaschun, G. Compactness of protein molten globules: temperature-induced structural changes of the apomyoglobin folding intermediate. Eur Biophys J. 1994;23(4):297–305. [PubMed]
Gittis, AG; Stites, WE; Lattman, EE. The phase transition between a compact denatured state and a random coil state in staphylococcal nuclease is first-order. J Mol Biol. 1993 Aug 5;232(3):718–724. [PubMed]
Griko, YV; Freire, E; Privalov, PL. Energetics of the alpha-lactalbumin states: a calorimetric and statistical thermodynamic study. Biochemistry. 1994 Feb 22;33(7):1889–1899. [PubMed]
Griko, YV; Gittis, A; Lattman, EE; Privalov, PL. Residual structure in a staphylococcal nuclease fragment. Is it a molten globule and is its unfolding a first-order phase transition? J Mol Biol. 1994 Oct 14;243(1):93–99. [PubMed]
Hagihara, Y; Tan, Y; Goto, Y. Comparison of the conformational stability of the molten globule and native states of horse cytochrome c. Effects of acetylation, heat, urea and guanidine-hydrochloride. J Mol Biol. 1994 Apr 1;237(3):336–348. [PubMed]
Hamada, D; Hoshino, M; Kataoka, M; Fink, AL; Goto, Y. Intermediate conformational states of apocytochrome c. Biochemistry. 1993 Oct 5;32(39):10351–10358. [PubMed]
Hamada, D; Kidokoro, S; Fukada, H; Takahashi, K; Goto, Y. Salt-induced formation of the molten globule state of cytochrome c studied by isothermal titration calorimetry. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10325–10329. [PubMed]
Hamada, D; Kuroda, Y; Kataoka, M; Aimoto, S; Yoshimura, T; Goto, Y. Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c. J Mol Biol. 1996 Feb 16;256(1):172–186. [PubMed]
Hughson, FM; Wright, PE; Baldwin, RL. Structural characterization of a partly folded apomyoglobin intermediate. Science. 1990 Sep 28;249(4976):1544–1548. [PubMed]
Kataoka, M; Hagihara, Y; Mihara, K; Goto, Y. Molten globule of cytochrome c studied by small angle X-ray scattering. J Mol Biol. 1993 Feb 5;229(3):591–596. [PubMed]
Kataoka, M; Head, JF; Persechini, A; Kretsinger, RH; Engelman, DM. Small-angle X-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly alpha-helical conformation. Biochemistry. 1991 Feb 5;30(5):1188–1192. [PubMed]
Kataoka, M; Goto, Y. X-ray solution scattering studies of protein folding. Fold Des. 1996;1(5):R107–R114. [PubMed]
Konno, T; Kataoka, M; Kamatari, Y; Kanaori, K; Nosaka, A; Akasaka, K. Solution X-ray scattering analysis of cold- heat-, and urea-denatured states in a protein, Streptomyces subtilisin inhibitor. J Mol Biol. 1995 Aug 4;251(1):95–103. [PubMed]
Kuroda, Y; Kidokoro, S; Wada, A. Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process. J Mol Biol. 1992 Feb 20;223(4):1139–1153. [PubMed]
Kuwajima, K. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins. 1989;6(2):87–103. [PubMed]
Kuwajima, K. The molten globule state of alpha-lactalbumin. FASEB J. 1996 Jan;10(1):102–109. [PubMed]
Kuwajima, K; Hiraoka, Y; Ikeguchi, M; Sugai, S. Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin. Biochemistry. 1985 Feb 12;24(4):874–881. [PubMed]
Kuwajima, K; Nitta, K; Yoneyama, M; Sugai, S. Three-state denaturation of alpha-lactalbumin by guanidine hydrochloride. J Mol Biol. 1976 Sep 15;106(2):359–373. [PubMed]
Lattman, EE; Fiebig, KM; Dill, KA. Modeling compact denatured states of proteins. Biochemistry. 1994 May 24;33(20):6158–6166. [PubMed]
Loh, SN; Kay, MS; Baldwin, RL. Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5446–5450. [PubMed]
Nishii, I; Kataoka, M; Goto, Y. Thermodynamic stability of the molten globule states of apomyoglobin. J Mol Biol. 1995 Jul 7;250(2):223–238. [PubMed]
Nishii, I; Kataoka, M; Tokunaga, F; Goto, Y. Cold denaturation of the molten globule states of apomyoglobin and a profile for protein folding. Biochemistry. 1994 Apr 26;33(16):4903–4909. [PubMed]
Olah, GA; Trakhanov, S; Trewhella, J; Quiocho, FA. Leucine/isoleucine/valine-binding protein contracts upon binding of ligand. J Biol Chem. 1993 Aug 5;268(22):16241–16247. [PubMed]
Peng, ZY; Kim, PS. A protein dissection study of a molten globule. Biochemistry. 1994 Mar 1;33(8):2136–2141. [PubMed]
Privalov, PL. Intermediate states in protein folding. J Mol Biol. 1996 May 24;258(5):707–725. [PubMed]
Ptitsyn, OB. Molten globule and protein folding. Adv Protein Chem. 1995;47:83–229. [PubMed]
Schulman, BA; Redfield, C; Peng, ZY; Dobson, CM; Kim, PS. Different subdomains are most protected from hydrogen exchange in the molten globule and native states of human alpha-lactalbumin. J Mol Biol. 1995 Nov 10;253(5):651–657. [PubMed]
Shortle, D; Abeygunawardana, C. NMR analysis of the residual structure in the denatured state of an unusual mutant of staphylococcal nuclease. Structure. 1993 Oct 15;1(2):121–134. [PubMed]
Sosnick, TR; Mayne, L; Hiller, R; Englander, SW. The barriers in protein folding. Nat Struct Biol. 1994 Mar;1(3):149–156. [PubMed]
Sosnick, TR; Trewhella, J. Denatured states of ribonuclease A have compact dimensions and residual secondary structure. Biochemistry. 1992 Sep 8;31(35):8329–8335. [PubMed]
Xie, D; Bhakuni, V; Freire, E. Calorimetric determination of the energetics of the molten globule intermediate in protein folding: apo-alpha-lactalbumin. Biochemistry. 1991 Nov 5;30(44):10673–10678. [PubMed]