The effects on protein stability of negatively charged Glu side chains at different positions along the length of the alpha-helix were investigated in the two-stranded alpha-helical coiled-coil. A native coiled-coil has been designed which consists of two identical 35 residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow the formation of an interchain 2-2' disulphide bridge. This coiled-coil contains no intra- or interchain electrostatic interactions and served as a control for peptides in which Glu was substituted for Gln in the e or g heptad positions. The effect of the substitutions on stability was determined by urea denaturation at 20 degrees C with the degree of unfolding monitored by circular dichroism spectroscopy. A Glu substituted for Gln near the N-terminus in each chain of the coiled-coil stabilizes the coiled-coil at pH 7, consistent with the charge-helix dipole interaction model. This stability increase is modulated by pH change and the addition of salt (KCl or guanidine hydrochloride), confirming the electrostatic nature of the effect. In contrast, Glu substitution in the middle of the helix destabilizes the coiled-coil because of the lower helical propensity and hydrophobicity of Glu compared with Gln at pH7. Taking the intrinsic differences into account, the apparent charge-helix dipole interaction at the N-terminus is approximately 0.35 kcal/mol per Glu substitution. A Glu substitution at the C-terminus destabilizes the coiled-coil more than in the middle owing to the combined effects of intrinsic destabilization and unfavourable charge-helix dipole interaction with the negative pole of the helix dipole. The estimated destabilizing charge-helix dipole interaction of 0.08 kcal/mol is smaller than the stabilizing interaction at the N-terminus. The presence of a 2-2'disulphide bridge appears to have little influence on the magnitude of the charge-helix dipole interactions at either end of the coiled-coil.

Amino Acid Sequence↗

PubMedDate not supplied

Lactam bridge stabilization of alpha-helical peptides: ring size, orientation and positional effects.

A series of 14 residue amphipathic alpha-helical peptides, in which the sidechains of glutamic acid and lysine have been covalently joined, was synthesized in order to determine the effect of spacing, position and orientation of these lactam bridges. It was found that although an (i, i+3) spacing would position the lactam bridge on the same face of the helix, these lactams with 18-member rings were actually helix-destabilizing regardless of position or location. On the other hand, (i, i+4) lactams with 21-member rings were helix-stabilizing but this was dependent on orientation. Glutamic acid-lysine lactams increased the helical content of the peptide when compared with their linear homologue in benign conditions (50 mM KH2PO4, 100 mM KCl, pH 7). Two Glu-Lys (i, i+4) lactams located at the N- and C-termini gave rise to a peptide with greater than 99% helical content in benign conditions. Peptides with Lys-Glu oriented lactams were random structures in benign conditions but in the presence of 50% TFE could be induced into a helical conformation. The stability of the single-stranded alpha-helices, as measured by thermal denaturations in 25% TFE indicated that Glu-Lys oriented lactam bridges stabilized the helical conformation relative to the linear unbridged peptide. One Glu-Lys lactam in the middle of the peptide was more effective at stabilizing helical structure than two Glu-Lys lactams positioned one at each end of the molecule. The lactams with the Lys-Glu orientation were destabilizing relative to the unbridged peptide. This study demonstrates that correct orientation and position of a lactam bridge is critical in order to design peptides with high helical content in aqueous media.

Amino Acid Sequence↗

PubMedDate not supplied

Relationship of sidechain hydrophobicity and alpha-helical propensity on the stability of the single-stranded amphipathic alpha-helix.

The aim of the present investigation is to determine the effect of alpha-helical propensity and sidechain hydrophobicity on the stability of amphipathic alpha-helices. Accordingly, a series of 18-residue amphipathic alpha-helical peptides has been synthesized as a model system where all 20 amino acid residues were substituted on the hydrophobic face of the amphipathic alpha-helix. In these experiments, all three parameters (sidechain hydrophobicity, alpha-helical propensity and helix stability) were measured on the same set of peptide analogues. For these peptide analogues that differ by only one amino acid residue, there was a 0.96 kcal/mole difference in alpha-helical propensity between the most (Ala) and the least (Gly) alpha-helical analogue, a 12.1-minute difference between the most (Phe) and the least (Asp) retentive analogue on the reversed-phase column, and a 32.3 degrees C difference in melting temperatures between the most (Leu) and the least (Asp) stable analogue. The results show that the hydrophobicity and alpha-helical propensity of an amino acid sidechain are not correlated with each other, but each contributes to the stability of the amphipathic alpha-helix. More importantly, the combined effects of alpha-helical propensity and sidechain hydrophobicity at a ratio of about 2:1 had optimal correlation with alpha-helix stability. These results suggest that both alpha-helical propensity and sidechain hydrophobicity should be taken into consideration in the design of alpha-helical proteins with the desired stability.

Amino Acids↗

PubMedDate not supplied

Design, synthesis and structural characterization of model heterodimeric coiled-coil proteins.

We report the design and synthesis of model heterodimeric coiled-coil proteins and the packing contribution of interchain hetero-hydrophobic side-chains to coiled-coil stability. The heterodimeric coiled-coils are obtained by oxidizing two 35-residue polypeptide chains, each containing a cysteine residue at position 2 and differing in amino acid sequences in the hydrophobic positions ("a" and "d") responsible for the formation and stabilization of the coiled-coil. In each peptide, a single Ala residue was substituted for Leu at position "a" or "d". The formation and stability of heterodimeric coiled-coils were investigated by circular dichroism studies in the presence and absence of guanidine hydrochloride and compared to the corresponding homodimeric coiled-coils. The coiled-coil proteins with an Ala substitution at position "a" were less stable than those with an Ala substitution at position "d" in both the homodimeric (Ala-Ala interchain interactions) and heterodimeric (Leu-Ala interchain interactions ) coiled-coils. The 70-residue disulfide bridged peptides (homo- and heterodimeric coiled-coils) can be readily separated by reversed-phase chromatography (RPC) even though they have identical amino acid compositions as well as in the hydrophobic "a" and "d" positions. The elution of the 70-residue peptides prior to their corresponding 35-residue monomers suggests that these proteins are retaining a large portion of their coiled-coil structure during RPC at pH2 and their retention behavior correlates with protein stability.

Amino Acid Sequence↗

PubMedDate not supplied

Tyrosine and tyrosinate fluorescence of S-100b. A time-resolved nanosecond fluorescence study. The effect of pH, Ca(II), and Zn(II).

The properties of the tyrosine and tyrosinate emissions from brain S-100b have been studied by nanosecond time-resolved fluorescence at emission wavelengths in the range 305 to 365 nm. The effect of pH on the fluorescence has been studied at pH 6.5, 7.5, and 8.5 for the Ca(II) apo and holo forms of the protein, and for the apo and holo forms in the presence and absence of Zn(II) at pH 7.5. The fluorescence decay is biexponential at pH 8.5 and triexponential at pH 6.5 and 7.5. The three components of the decay have wavelength and metal ion dependent lifetimes in the ranges 0.06 to 1.05 ns, 0.49 to 3.76 ns, and 3.60 to 14.5 ns. The observation of a long lifetime component at wavelengths characteristic of emission from tyrosinate suggests that in class A proteins this may be a useful diagnostic of the environment of tyrosine in their native structures. The time-resolved emission spectra provide evidence for efficient, subnanosecond protolysis of the excited state of the single tyrosine (Tyr17) under all conditions studied except in 6 M guanidium chloride in which the protein shows only emission from tyrosine (lambda em 305 nm), suggesting that the tyrosinate emission is a property of the tertiary structure of the native protein. The Zn(II)-dependence of the fluorescence is fully consistent with the earlier suggestion that Tyr17 is near the Zn(II) binding site and remote from the high affinity Ca(II) binding site.

Animals↗

PubMedDate not supplied

Synthetic model proteins: contribution of hydrophobic residues and disulfide bonds to protein stability.

The objective of this study was to evaluate quantitatively the contribution that hydrophobic residues and disulfide bonds make to protein stability using a model protein with well-defined secondary, tertiary and quaternary structure. This de novo-designed protein consists of two identical 35-residue alpha-helical polypeptide chains arranged in a coiled-coil structure, which are stabilized by nine pairs of interchain hydrophobic interactions from leucine residues and an interchain disulfide bond. The mutant proteins differ from the Leu-protein only at positions 16 and 19 of each chain which contain either Ile, Val, Ala, Phe or Tyr instead of Leu residues. The stability of each protein was determined in its reduced and oxidized form from guanidine hydrochloride denaturation experiments using circular dichroism measurements. The Leu-, Ile-, Val- and Phe-proteins containing the disulfide bond were essentially 100% alpha-helical in benign medium (0.05 M phosphate buffer, pH 7, containing 0.1 M KCl) with transition midpoints of 5.3, 4.1, 2.9 and 2.4 M denaturant, respectively. Reduction of the disulfide bond had little effect on the Leu-protein but resulted in significant decreases in helicity of the other mutants. The order of protein stability of these analogs remains the same in the absence or presence of the disulfide bond (Leu- greater than Ile- greater than Val- greater than Phe- greater than Tyr- greater than Ala-protein). Interestingly, the more stable the protein was in the absence of the disulfide bond, the larger the contribution the disulfide bond made to protein stability. The identical mutation in the disulfide bridge protein had a much greater effect on protein stability than in the reduced protein. This suggests that the disulfide bond controls the conformation of the coiled-coil some 14-17 residues further along the polypeptide chain (the disulfide bond is between positions 2 and 2' and the closest mutation site in the coiled-coil is at position 16 and 16'). In contrast, in the absence of the disulfide bond, the coiled-coil is more flexible and can accommodate the mutations more easily by adjusting the interchain packing around the mutation sites. The helix-forming tendency of nonpolar residues was deduced in this study by comparing the alpha-helicity of the mutant polypeptides in aqueous buffer containing 50% trifluoroethanol where they exist as single-stranded helices. Hydrophobicity is an important factor in determining the alpha-helix forming tendency of aliphatic nonpolar residues in amphipathic alpha-helices.

Amino Acid Sequence↗

PubMedDate not supplied

Protein design using model synthetic peptides.

We have designed and synthesized a small, unique protein molecule with defined secondary, tertiary and quaternary structure. This 35-residue peptide, containing a cysteine residue at its N-terminal end, was oxidized to form a 70-residue disulfide-linked two-stranded alpha-helical coiled-coil with the two alpha-helices parallel and in-register. The major contribution to the formation and stabilization of the alpha-helical coiled-coil is hydrophobic interactions between positions 2 and 5 of the heptapeptide repeat (Lys-Leu-Glu-Ala-Leu-Glu-Gly). The protein (L-protein) contains nine leucine-leucine hydrophobic interactions between the alpha-helices of the coiled-coil. Circular dichroism studies demonstrated that this protein in its reduced ([L (r)] or oxidized (L (o)] state was essentially 100% alpha-helical ([theta]220 = -34,050 and -32,000 degrees respectively) at pH 2 (0.1% aqueous trifluoroacetic acid). Our objective was to modify systematically the structure of L to delineate the contribution that various amino acid side chains make to the formation and stabilization of its three-dimensional structure. A-protein, which contains alanine instead of leucine at positions 16 and 19 of the hydrophobic repeat in each chain of the coiled-coil, was compared to the L-protein. At pH 2, the oxidized form of the A-protein [A (o)] was essentially 100% helical. However, the protein was much less stable to temperature denaturation compared to the L-protein. The replacement of two leucine-leucine interactions by two alanine-alanine interactions has a dramatic effect on the formation and stability of the two-stranded alpha-helical coiled-coil structure. The results of this study clearly demonstrate the validity of this synthetic model protein approach to understanding the molecular aspects responsible for the folding and stabilization of protein molecules.

Amino Acid Sequence↗