Specific Sequences Determine the Stability and Cooperativity of Folding of the C-terminal Half of Tropomyosin

Abstract

208 circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of ureainduced unfolding. Temperature and urea denaturation experiments show that Tm 143-235 is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of ␤-branched residues located at d positions.The coiled-coil motif mediates the process of oligomerization of many proteins. This structure is a consequence of a heptapeptide repetition (abcdefg) in the chemical nature of the residues in the primary structure of the polypeptide chain (1-3). An ␣-helical conformation places hydrophobic residues at positions a and d on the same side of the helix, creating a nonpolar interface that promotes dimerization through the burial of hydrophobic surface. Residues at positions e and g are often charged and may form salt bridges with residues at positions g and e, respectively, of the other helix. The maximization of favorable ionic attractions and the minimization of unfavorable repulsions probably influence the particular alignment of the ␣-helical chains (4). The folding of synthetic peptides that adopt a coiled-coil structure has been investigated extensively (4 -11). Synthetic peptide models for coiled-coils may not always be taken as representative because they often employ regular patterns of residues at positions a and d and lack i, iϩ3 and i, iϩ4 intrahelical ionic interactions (7,12). Natural coiled-coil sequences, such as tropomyosin (Tm), 1 are more complex, with irregular patterns of intrahelix and interhelix side chain-side chain interactions and a greater variety of residues at the hydrophobic core. Tm can be used as a natural model for the study of the principal interactions that maintain the coiled-coil structure and ␣-helix stability.Muscle ␣-Tm is a symmetric coiled-coil composed of two parallel and in register ϳ410 Å, 284-residue ␣-helices (2, 13, 14). The C-terminal half of the Tm molecule is less thermally stable than the N-terminal half (15,16). A series of studies (17,18) have suggested that Tm fragments with less than 94 residues were unable to form stable secondary structures at low micromolar concentrations. Recently, Holtzer et al. (19) showed that a 65-amino acid fragment (residues 190 -254) presents a significant amount of ␣-helix (ϳ43%) when present in high micromolar concentrations (115 M) at 10°C but is essentially unfolded at 25°C.Microcalorimetric analysis of ␣-Tm thermal denaturation has been interpreted as a multistep process in which spec...

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

“…In the absence of Mg 21 , the presence of 100 mM NaCl caused a decrease in the cleavage rate of Tm by chymotrypsin. These results are consistent with previous observations that Tm is more stable in high salt conditions [35][36][37][38][39] and that…”

“…Although this peptide is less stable than longer C-terminal Tm fragments, 35,41 it has been shown to interact with a Tm N-terminal fragment to form the head-to-tail complex. 38 Initially, we 21 did not induce protein aggregation in the absence of 100 mM NaCl; this allowed us to obtain fully reversible thermal denaturation curves.…”

“…It may be expected that repulsive interactions between those residues could create regions of local instability in the protein. Indeed, the a-helical conformation of full-length Tm or its fragments are stabilized at high ionic strength conditions, [35][36][37][38] which possibly increases the stability of the coiledcoil by reducing charge-charge and dipole-dipole repulsive interactions while at the same time enhancing apolar-apolar interactions. 37 In a previous study, we showed that the addition of Mg 21 induced a remarkable increase in the thermal stability of two fragments corresponding to the N-and Cterminal halves of Tm, 15 suggesting that Mg 21 binds to both fragments.…”

Mapping of the Lipid-Binding and Stability Properties of the Central Rod Domain of Human Dystrophin

“…Thus, the molar ellipticity at 222 nm remains unchanged during an inter-helix interaction, while a decrease is observed in the molar ellipticity at 208 nm. [20][21][22][23] Zhou et al determined empirically that the θ 222 /θ 208 ratio is equal to or higher than 1 for two-stranded coiled coils, whereas for noninteracting helices, the ratio is lower than 1, lying between 0.8 and 0.9. 24 In our study, the θ 222 /θ 208 ratios range from 1.02 to 1.15 for the six well-folded proteins (Fig.…”

Ca2+-induced Rolling of Tropomyosin in Muscle Thin Filaments

“…Proteins-Tryptophan codons were created at positions 90, 94, 101, 107, 111, 122, 261, 276, and 278 of the chicken fast skeletal Tm cDNA with the dipeptide Ala-Ser N-terminal fusion (ASTm) (32) by PCRbased oligonucleotide-mediated site-directed mutagenesis as described (33)(34)(35), and the mutated cDNAs were transferred to the pET-3a expression vector (36). The original residues in the chicken skeletal Tm that were mutated are as follows: Arg-90, Leu-94, Arg-101, Ala-107, Gln-111, Glu-122, Tyr-261, His-276, and Leu-278.…”