Alfred Holtzer

Theory of α-helix-to-random-coil transition of two-chain, coiled coils. Application of the augmented theory to thermal denaturation of ββ tropomyosin

The statistical mechanical theory of the helix-to-random-coil transition in two-chain, a-helical coiled coils has recently been augmented by inclusion of the effects of loop entropy and out-of-register (“mismatched”) structures. This theory is applied to experimental data on non-cross-linked a-tropomyosin at nearly neutral and at acidic pH, using extant values of helix initiation (u) and propagation (s) parameters for each amino acid in the sequence. A semiquantitative fit of the helix content (from circular dichroism measurements) vs. temperature (0-80 “C) is obtained at each pH, covering a 1000-fold range of protein concentration. The algorithms for the mean interhelix interaction free energy per mole of turn pairs (RT In w(T)) needed to produce the fit at each pH provide curves of RT In w ( T ) vs. T that are similar in range and in shape, each showing a minimum near room temperature. Theory is also compared with independent experiments, in particular light scattering and cross-linkability studies at nearly neutral pH. The temperature dependence of the weight-average molecular weight at nearly neutral pH, as recently determined by light scattering, agrees well with the theoretical prediction. The observed high degree of cross-linkability of tropomyosin in the native state can be reconciled with the theoretically calculated fraction of in-register molecules under the benign conditions of the cross-linking experiments. Examination shows that the principal cause of the greater stability of a-tropomyosin at low pH lies in the augmented short-range (a,s) interactions of aspartic and glutamic residues over those of the aspartate and glutamate species which predominate near neutral pH. In fact, it is shown that, with small adjustment (within experimental error) in these parameters, the same interhelix interaction free energy algorithm can be used to explain the full range of data at both pHs. A discussion of the implications of this result is given, wherein it is shown that the interhelix salt bridges, while they may provide enough free energy at nearly neutral pH to ensure that the helices associate in parallel, make a contribution to the total interhelix interaction that is relatively small compared with the hydrophobic contribution. The statistical theory developed here is compared with the all-or-none-stages model brought forward elsewhere; it is suggested that the latter disagrees with the recent light scattering data and is difficult to reconcile with accepted ideas concerning loop entropy.

The effect of various monovalent anions on myosin B solutions. The identification of actin as a product of ATP action.

Abstract Physical studies on Myosin B solutions in KSCN, KI, and in a variety of other ionic media reveal that exposure to these media leads to a dissociation into myosin and actin moieties. The actin appears in the globular form and may be clearly identified in the ultracentrifuge. The peak formed under these conditions (G-actin) is indistinguishable from the peaks formed in the same media by conventionally prepared actin or by redissolution of the pellet obtained by addition of ATP to Myosin B and strong centrifugation. It is concluded that actin exists in Myosin B in combination with myosin but otherwise in substantially the same form as is obtained by conventional procedures, employing treatment with non-aqueous solvents; and that actin is indeed a product produced by the action of ATP on Myosin B.

CD and13C?-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of ?-tropomyosin

Synthesis and CD and (13)C(alpha)-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide ((190)Tm(254)) comprising residues 190-254 of the alpha-tropomyosin chain. CD on a version disulfide cross-linked via the N-terminal cysteine side chains indicates that this dimer is highly helical and melts near 48 degrees C. The CD is independent of peptide concentration, showing that association of (190)Tm(254) stops at the two-strand level. Similar studies on the reduced version show much lower helix content at low temperature, melting points below room temperature, and the expected concentration dependence. The observed melting temperature of the reduced peptide is far below (by 27 degrees C) that expected from an extant analysis of calorimetry data on parent tropomyosin that designates (190)Tm(254) as an independently melting cooperative block. This disagreement and the pronounced nonadditivity seen when data for (190)Tm(254) are combined with extant data for other subsequences argue decisively against the concept of specific independently melting blocks within the tropomyosin chain. The data for (190)Tm(254) also serve to test recent ideas on the sequence determinants of structure and stability in coiled coils. Analysis shows that some ideas, such as the stabilizing effect of leucine in the d heptad position, find support, but others--such as the destabilizing effect of alanine in d, the dimer-disfavoring effect of beta-branching in d and its dimer-favoring effect in a, and the dimer-directing effect of asparagine in a--are more questionable in tropomyosin than in the leucine zipper coiled coils. (13)C(alpha)-NMR data at two labeled sites, L228(d) and V246(a), of (190)Tm(254) display well-separated resonances for folded and unfolded forms at each site, indicating that the transition is slow on the NMR time scale and thus demonstrating the possibility of obtaining thermodynamic and kinetic information on the transition at the residue level.