Domain characteristics of the carboxyl‐terminal fragment 206–316 of thermolysin: pH and ionic strength dependence of conformation

Abstract

SynopsisThe pH and ionic strength dependence of conformation of the COOH-terminal fragment 206-316 (fragment FII) of thermolysin was monitored by far-uv CD and difference absorption measurements. This fragment was shown previously to possess the properties of a protein domain, i.e., able to refold into a stable nativelike structure [Fontana, A., attained at neutral pH). Quantitative analysis of secondary structure from CD spectra revealed that state H at 4°C is characterized by some 30% a-helical structure, compared to 47% for state N. The heat-and Gdn -HC1-mediated unfolding transitions of state H were fully reversible and characterized by little cooperativity, which is taken as an indication that state H corresponds to several species possessing different, and low, conformational stabilities. The midpoint transition from state H to N occurs near pH 2.5, implying that the acid transition results from the titration of carboxyl groups of the fragment with anomalously low pK, as would be expected for groups involved in specific salt bridges. Fragment FII at pH 1 (state H) may be induced to exhibit nearly the same degree of helicity of state N simply by increasing the ionic strength of the solution, thus reducing the repulsive interactions between positive charges within the highly charged fragment at pH 1. The results obtained emphasize the role of electrostatic interactions in the folding and stability of fragment FII and suggest a mechanism of folding of the fragment from U to N involving an intermediate state characterized by an assembly of fluctuating a-helices.

Classification of Acid Denaturation of Proteins: Intermediates and Unfolded States

“…In these cases, upon decreasing the pH, the protein will usually go directly into the molten globule state from the native state. There are several reports in the literature that indicate that other proteins not examined in this study also form compact intermediate states at low pH: examples include interferon (Arakawa et al, 1987), carmín (Rao & Prakash, 1989, the C-terminal fragment of thermolysin (Dalzoppo et al, 1985), interleukin-2 (Dryden & Weir, 1991), interleukin-4 (Redfield etal., 1994), immunoglobulin (Buchner etal., 1991), retinol-binding protein (Bychkova etal., 1992), brain-derived neurotrophic factor (Narhi et al, 1993), transthyretin (Colon & Kelly, 1992), 0-globulin (Rajendran & Prakash, 1992), BSA (Tanford et al, 1955), and growth hormone (Holzman etal., 1990). In addition, similar phenomena may be observed at alkaline pH (McPhie, 1982;Goto & Fink, 1989).…”

CD and 1H‐NMR studies on the conformational properties of peptide fragments from the C‐terminal domain of thermolysin

“…This is because protein fragments when isolated are suitable models of potential nucleation centers, which, in the absence of tertiary interactions, may direct the folding process through diffusion-collision events. Factors affecting the final stability of protein fragments, when incorporated into the native structure, can also be suitably investigated by analyzing their propensity to form secondary structures under different conditions of pH, temperature, ionic strength, denaturing agents and mixed solvents (Baldwin, 1 990).The C-terminal fragment 206 -31 6 of thermolysin, a protease from Bacillus thermoproteoliticus (Endo, 1962), has been characterized by Fontana's group (Dalzoppo et al, 1985a;Vita et al, 1982) as a protein domain, lacking disulfide bonds, able to fold independently from the rest of the protein. Even the shorter fragment 255-316 (a single chain of 62 residues) folds independently into a compact and very Correspondence to M. Rico, Instituto de Estructura de la Materia, Consejo Superior de Investigaciones Cientificas, Serrano 119, E-28006 Madrid, SpainAbbreviations.…”

“…The C-terminal fragment 206 -31 6 of thermolysin, a protease from Bacillus thermoproteoliticus (Endo, 1962), has been characterized by Fontana's group (Dalzoppo et al, 1985a;Vita et al, 1982) as a protein domain, lacking disulfide bonds, able to fold independently from the rest of the protein. Even the shorter fragment 255-316 (a single chain of 62 residues) folds independently into a compact and very Correspondence to M. Rico, Instituto de Estructura de la Materia, Consejo Superior de Investigaciones Cientificas, Serrano 119, E-28006 Madrid, Spain…”

Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of .alpha.-lactalbumin and lysozyme

“…Although chemical trapping is an effective approach to study the folding reactions accompanied by disulfide intermediates (Creighton, 1978;Creighton & Goldenberg, 1984), the effects of the chemical groups, introduced by the trapping reaction, on protein structure cannot be neglected (Goto & Hamaguchi, 1979; Chavez & Scheraga, 1980; White, 1982) [see also Kosen et al (1983)] and very likely interfere with the structural characterization of the intermediates. One way out of this difficulty is to find out proteins that show stable structural intermediates during the equilibrium unfolding reactions so that the structures of the intermediates can be characterized at equilibrium (Wong & Tanford, 1973;Robson & Pain, 1976; Kuwajima, 1977; Adams et al, 1980;McCoy et al, 1980;Dolgikh et al, 1984;Mitchinson & Pain, 1985;Dalzoppo et al, 1985). However, general significance of such apparently exceptional cases has not yet been established, and a stringent test of identity of the This work was supported in part by Grants-in-Aid for Scientific Research [General Research (c), 58580200 and 60580217] from the Ministry of Education, Science and Culture of Japan.…”