The molten globule intermediate of apomyoglobin and the process of protein folding

Abstract

The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of a-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N e I e U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of 1, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagenesis experiments indicate that the interactions between the A, G, and H helices in 1 are loose (Hughson, EM., Barrick, D., & Baldwin, R.L., 1991, Biochemistry30, 4113-41 18), which can explain why I is formed rapidly from U at the start of folding. These experiments are consistent with the explanation proposed earlier (Baldwin, R.L., 1989, Trends Biochem. Sci. 14, 291-294) for the stabilization of native secondary structures in molten globule intermediates, namely that each unit of native secondary structure has a hydrophobic face, and the hydrophobic surfaces of two units of secondary structure can interact loosely to provide mutual stabilization. These topics are discussed here in the light of some additional results.Keywords: apomyoglobin; energetics of folding; folding pathways; molten globule; protein folding It is now generally agreed that structured intermediates are observable during the kinetics of folding of most small proteins. If they are on-pathway intermediates, then it is possible to determine the folding pathway by characterizing their structures. The method of pulsed H/D exchange, which measures the exchange rates of individual peptide NH protons in a folding intermediate using the Abbreviations: Mb, myoglobin; apoMb, apomyoglobin; holoMb, holomyoglobin; 2D, two-dimensional; H-bond, hydrogen bond; H/D, hydrogenldeuterium; ANS, 1-anilinonaphthalene-8-sulfonic acid; or-LA, a-lactalbumin; cyt c, cytochrome c; pH,, pH midpoint of an unfolding transition; pH', pH measured in *HZO without correction for the isotope effect; GdmC1, guanidinium chloride.

The Structure of Denatured α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

“…Under a variety of conditions, ␣-LA adopts a partially structured conformation termed as "molten globule" (1)(2)(3)(4)(5)(6)(7)(8)(9), which has been observed along both the unfolding (1, 2, 5, 10 -12) and folding pathways (13)(14)(15)(16)(17)(18) of ␣-LA and is considered one of the best characterized folding/unfolding intermediates of globular proteins. At low pH (1,19,20), elevated temperature (19,21), or mild concentration of denaturant (e.g.…”

“…Like most denatured or partially denatured state of proteins (30,31), it comprises a large number of conformational isomers. So far, characterization of the structure of denatured ␣-LA and the molten globule state of ␣-LA have been achieved by measuring the average property of these collective isomers using a wide range of spectroscopic and physiochemical methods, including circular dichroism (1,2,19,32), fluorescence (12,32,33), NMR (5,14,15,20,29,34), disulfide replacement (35)(36)(37), limited proteolysis (38,39), light scattering (40,41), and calorimetric techniques (42). Further understanding of the denatured state of ␣-LA and the molten globule state of ␣-LA will require fractionation of diverse populations of conformational isomers that constitute the denatured ␣-LA.…”

The Cytochrome c Fold Can Be Attained from a Compact Apo State by Occupancy of a Nascent Heme Binding Site

“…Small structural differences have been identified between the two redox forms of cytochrome c (25,26). In addition it has been suggested that phosphate binding to lysine-rich sites at low ionic strengths in the oxidized state may contribute to the increase in the effec- 1 The abbreviation used is: ANS, 8-anilino-1-napthalenesulphonate. .…”

“…see Refs. [1][2][3]. Such studies are of particular significance for the c-type class of cytochromes where the heme group must become covalently bound to the protein to generate the functional native structure (4).…”

Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation

“…For many years this equilibrium assumption, which is valid in many instances, contributed to the misunderstanding of folding pathways, which are now recognized to quite clearly have multiple kinetic intermediates (Kim & Baldwin, 1982;Barrick & Baldwin, 1993).…”