The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin.

We have used a synthetic peptide consisting of the first 30 residues of striated muscle alpha-tropomyosin, with GlyCys added to the C-terminus, to investigate the effect of N-terminal acetylation on the conformation and stability of the N-terminal domain of the coiled-coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low alpha-helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N-terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2-chained coiled coil at high ionic strength. Disulfide crosslinking and N-terminal acetylation both greatly stabilized the coiled-coil alpha-helical conformation in high salt. Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its alpha-helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled-coil formation. In the presence of the organic solvents, N-terminal acetylation had very little effect on the conformation or stability of the peptide. Our results indicate that N-terminal acetylation stabilizes coiled-coil formation in the peptide. The effect cannot be explained by interactions with the "helix-dipole" because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N-terminal acetylation has only small effects on the overall stability of tropomyosin.     

Recombinant small subunit of smooth muscle myosin light chain phosphatase. Molecular properties and interactions with the targeting subunit.

We expressed the small subunit of smooth muscle myosin light chain phosphatase (MPs) in Escherichia coli, and have studied its molecular properties as well as its interaction with the targeting subunit (MPt). MPs (M(r) = 18,500) has an anomalously low electrophoretic mobility, running with an apparent M(r) of approximately 21,000 in sodium dodecyl sulfate-gel electrophoresis. CD spectroscopy shows that it is approximately 45% alpha-helix and undergoes a cooperative temperature-induced unfolding with a transition midpoint of 73 degrees C. Limited proteolysis rapidly degrades MPs to a stable C-terminal fragment (M(r) = 10,000) that retains most of the helical content. Rotary shadowing electron microscopy reveals that it is an elongated protein with two domains. Sedimentation velocity measurements show that recombinant MPt (M(r) = 107,000), intact MPs, and the 10-kDa MPs fragment are all dimeric, and that MPs and MPt form a complex with a molar mass consistent with a 1:1 heterodimer. Sequence analysis predicts that regions in the C-terminal portions of both MPs and MPt have high probabilities for coiled coil formation. A synthetic peptide from a region of MPs encompassing residues 77-116 was found to be 100% alpha-helical, dimeric, and formed a complex with MPt with a molecular mass corresponding to a heterodimer. Based on these results, we propose that MPs is an elongated molecule with an N-terminal head and a C-terminal stalk domain. It dimerizes via a coiled coil interaction in the stalk domain, and interacts with MPt via heterodimeric coiled coil formation. Since other proteins with known regulatory function toward MP also have predicted coiled coil regions, our results suggest that these regulatory proteins target MP via the same coiled coil strand exchange mechanism with MPt.     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.     

A scanning calorimetric study of the thermally induced unfolding of various forms of tropomyosin

The reversible thermally induced unfolding of various forms of tropomyosin, a two-chain alpha-helical coiled coil, has been studied by high-sensitivity differential scanning calorimetry (DSC). Included in the study are the reduced and oxidized (disulfide cross-linked) forms of alpha alpha- and beta beta-tropomyosin, and the forms of alpha alpha-tropomyosin in which all sulfhydryl groups have been blocked by carboxymethylation or carboxyamidomethylation. Oxidation or blocking of the sulfhydryl groups of tropomyosin strongly affect the thermotropic behavior of the protein in unpredictable ways. The empirical results presented here are in qualitative agreement with those from an earlier DSC study of the oxidized and carboxymethylated forms of alpha alpha-tropomyosin [S.A. Potekhin and P.L. Privalov (1982) Journal of Molecular Biology, Vol. 159, pp. 519-535], but we find that a different decomposition into subtransitions is possible. Comparison of the alpha alpha and beta beta species indicates, in agreement with extant CD studies, that the noncross-linked beta beta species is somewhat less stable than its alpha alpha counterpart, but that cross-linking enhances the stability of the beta beta doubly cross-linked species by a greater amount and does not lead to the small low-temperature transition ("pretransition") seen in the singly cross-linked alpha alpha species.     

Kinetics of folding and unfolding of alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin.

Stopped flow CD (SFCD) kinetic studies of self-assembly of coiled coils of rabbit alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin (NPTm) are reported. The protein was denatured in 6 M urea buffer, then renatured by 10-fold dilution into benign saline buffer. Folding was monitored by SFCD in the backbone region (222 nm). Protein chains are shown to be totally unfolded (and separated in the reduced species) in the initial denaturing medium and fully folded as two-chain coiled coils in the final benign medium. In all cases of folding in benign buffer of totally unfolded chains, two phases were found in the folding process: a fast phase (less than 0.04 s, the SFCD dead time), in which an intermediate state with about 70% of the equilibrium ellipticity forms; followed by a slower, observable phase that completes the folding. The slow phase is first order (k-1 = 1.6 s at 20 degrees C), signifying that chain association for reduced samples occurs in the fast phase. In contrast, folding in benign buffer from an initial state with 70% of the equilibrium ellipticity is all fast, suggesting that the folding intermediate is not an equilibrium species. Cross-linking at Cys-190 increases the helix content of the fast-formed intermediate state to about 85% of the equilibrium value, but leaves the rate constant of the slow phase unchanged. In NPTm, which does not form high aggregates at low ionic strength, the rate of the observable phase is almost independent of ionic strength in the range of approximately 0.15-0.6 M, but is reduced one to two orders of magnitude by further reduction to 0.026 M. In folding from totally unfolded chains, the rate is reduced less than one order of magnitude by changing the final state to about 50% folded. In contrast to folding, unfolding of alpha alpha-tropomyosin from the native state is all fast.     

NMR studies of amyloid beta-peptides: proton assignments, secondary structure, and mechanism of an alpha-helix----beta-sheet conversion for a homologous, 28-residue, N-terminal fragment.

Beta-peptide is a major component of amyloid deposits in Alzheimer's disease. We report here a proton nuclear magnetic resonance (NMR) spectroscopic investigation of a synthetic peptide that is homologous to residues 1-28 of beta-peptide [abbreviated as beta-(1-28)]. The beta-(1-28) peptide produces insoluble beta-pleated sheet structures in vitro, similar to the beta-pleated sheet structures of beta-peptide in amyloid deposits in vivo. For peptide solutions in the millimolar range, in aqueous solution at pH 1-4 the beta-(1-28) peptide adopts a monomeric random coil structure, and at pH 4-7 the peptide rapidly precipitates from solution as an oligomeric beta-sheet structure, analogous to amyloid deposition in vivo. The NMR work shown here demonstrates that the beta-(1-28) peptide can adopt a monomeric alpha-helical conformation in aqueous trifluoroethanol solution at pH 1-4. Assignment of the complete proton NMR spectrum and the determination of the secondary structure were arrived at from interpretation of two-dimensional (2D) NMR data, primarily (1) nuclear Overhauser enhancement (NOE), (2) vicinal coupling constants between the amide (NH) and alpha H protons, and (3) temperature coefficients of the NH chemical shifts. The results show that at pH 1.0 and 10 degrees C the beta-(1-28) peptide adopts an alpha-helical structure that spans the entire primary sequence. With increasing temperature and pH, the alpha-helix unfolds to produce two alpha-helical segments from Ala2 to Asp7 and Tyr10 to Asn27. Further increases in temperature to 35 degrees C cause the Ala2-Asp7 section to become random coil, while the His13-Phe20 section stays alpha-helical. A mechanism involving unfavorable interactions between charged groups and the alpha-helix macrodipole is proposed for the alpha-helix----beta-sheet conversion observed at midrange pH.     

A calorimetric study of the folding-unfolding of an alpha-helix with covalently closed N and C-terminal loops.

The thermal melting of a dicyclic 29-residue peptide, having helix-stabilizing side-chain to side-chain covalent links at each terminal, has been studied by circular dichroism spectropolarimetry (CD) and differential scanning calorimetry (DSC). The CD spectra for this dicyclic peptide indicate that it is monomeric, almost fully alpha-helical at -10 degrees C, and undergoes a reversible transition from the folded to the disordered state with increasing temperature. The temperature dependencies of the ellipticity at 222 nm and the excess heat capacity measured calorimetrically are well fit by a two-state model, which indicates a cooperative melting transition that is complete within the temperature ranges of these experiments (from -10 degrees C to 100 degrees C). This allows a complete analysis of the thermodynamics of helix formation. The helix unfolding is found to proceed with a small positive heat-capacity increment, consistent with the solvation of some non-polar groups upon helix unfolding. It follows that the hydrogen bonds are not the only factors responsible for the formation of the alpha-helix, and that hydrophobic interactions are also playing a role in its stabilization. At 30 degrees C, the calorimetric enthalpy and entropy values are estimated to be 650(+/-50) cal mol(-1)and 2.0(+/-0.2) cal K(-1)mole(-1), respectively, per residue of this peptide. Comparison with the thermodynamic characteristics obtained for the unfolding of double-stranded alpha-helical coiled-coils shows that at that temperature the enthalpic contribution of non-polar groups to the stabilization of the alpha-helix is insignificant and the estimated transition enthalpy can be assigned to the hydrogen bonds. With increasing temperature, the increasing magnitude of the negative enthalpy of hydration of the exposed polar groups should decrease the helix-stabilizing enthalpy of the backbone hydrogen bonds. However, the helix-stabilizing negative entropy of hydration of these groups should also increase in magnitude with increasing temperature, offsetting this effect.     

Alpha-helix to random-coil transitions of two-chain coiled coils: the use of physical models in treating thermal denaturation equilibria of isolated subsequences of alpha alpha-tropomyosin

Two extant models of thermal folding/unfolding equilibria in two-chain, alpha-helical coiled coils are tested by comparison with experimental results on excised, isolated subsequences of rabbit alpha alpha-tropomyosin (Tm). These substances are designated iTmj where i and j are, respectively, the residue numbers (in the 284-residue parent chain) of the N- and C-terminal residues of the subsequence. One model postulates that a coiled coil consists of segments, each denaturing in an all-or-none manner, like small globular proteins. Thus this model yields a small number of populated molecular species. In an extant calorimetry study of 11Tm127 and of 190Tm284, each required only two all-or-none-segments, and their enthalpies and transition temperatures were assigned. These assignments are shown here to yield the concentration of all molecular species, and therefore the helix content, as a function of temperature. Such calculations for 190Tm284 are in tolerable agreement with CD experiments, but those for 11Tm127 are in gross disagreement. Thus, either the model itself or the calorimetric assignment is faculty. In the second model, all conformational states are counted and weighted, as in the Zimm-Bragg theory for single-chain polypeptides. This theory has been extended (by Skolnick) to two-chain coiled coils and is here used to fit CD data for 11Tm127, 142Tm281, and 190Tm284. The fit is tolerable for 11Tm127, good for 142Tm281, and quantitative for 190Tm284. Thus this comparison does not falsify this second model. The helix-helix interaction free energy, obtainable from the fit, shows nonadditivity when isolated subsequences are compared with the parent. This suggests that removal of a region from a long coiled coil allows energetically substantial adjustments in side-chain packing in the helix-helix interface. Thus, the helix-helix interaction in long coiled coils is characteristic of a global free energy minimum and not just of the regional constellation of side chains.     

Designed coiled-coil proteins: synthesis and spectroscopy of two 78-residue alpha-helical dimers.

Receptor-adhesive modular proteins are nongenetic proteins designed to contain ligand, spacer, coil, and linker modules and to interact strongly with integrins or other types of cell-surface receptors. We have designed, chemically synthesized, and characterized a 39-residue peptide chain having a 6-residue ligand module (Gly-Arg-Gly-Asp-Ser-Pro-) for adherence to Arg-Gly-Asp-binding integrin receptors, a 3-residue spacer module (-Gly-Tyr-Gly-) for flexibility, and a 30-residue coil module [-(Arg-Ile-Glu-Ala-Ile-Glu-Ala) 4-Arg-Cys-NH2] containing four 7-residue repeats for dimerization. This chain was designed to form a 78-residue noncovalent dimer (P39) by folding the coils of two chains into an alpha-helical coiled coil through hydrophobic interaction of eight pairs of Ile residues. Air oxidation of P39 gave P78, a 78-residue covalent dimer having a disulfide bridge linking its C termini. Raman spectroscopy indicated that both synthetic proteins have high alpha-helical content. Ultraviolet circular dichroic spectroscopy indicated that both dimers contain stable alpha-helical coiled coils. Its C-terminal disulfide bridge renders P78 significantly more stable than P39 to thermal denaturation or denaturation by urea. The coiled coil of P39 was 30% unfolded near 55 degrees C and half-unfolded in 8 M urea, while that of P78 was 30% unfolded only near 85 degrees C. These studies have demonstrated the feasibility of using these ligand, spacer, and coil modules to construct the designed coiled-coil proteins P39 and P78, a stage in the nanometric engineering of receptor-adhesive modular proteins.     

Thermal unfolding of myosin rod and light meromyosin: circular dichroism and tryptophan fluorescence studies

Rabbit skeletal myosin rod, which is the coiled-coil alpha-helical portion of myosin, contains two tryptophan residues located in the light meromyosin (LMM) portion whose fluorescence contributes 27% to the fluorescence of the entire myosin molecule. The temperature dependence of several fluorescence parameters (quantum yield, spectral position, polarization) of the rod and its LMM portion was compared to the thermal unfolding of the helix measured with circular dichroism. Rod unfolds with three major helix unfolding transitions: at 43, 47, and 53 degrees C, with the 43 and 53 degrees C transitions mainly located in the LMM region and the 47 degrees C transition mainly located in the subfragment 2 region. The fluorescence study showed that the 43 degrees C transition does not involve the tryptophan-containing region and that the 47 degrees C transition produces an intermediate with different fluorescence properties from both the completely helical and fully unfolded states. That is, although the fluorescence of the 47 degrees C intermediate is markedly quenched, the tryptophyl residues do not become appreciably exposed to solvent until the 53 degrees C transition. It is suggested that although the intermediate that is formed in the 47 degrees C transition contains an extensive region which is devoid of alpha-helix, the unfolded region is not appreciably solvated or flexible. It appears to have the properties of a collapsed nonhelical state rather than a classical random coil.     

Kinetics of folding and unfolding of beta beta-tropomyosin.

The kinetics of folding from random coils to two-chain coiled coils of beta beta-tropomyosin was studied by stopped-flow CD (SFCD) in the backbone region (222 nm). Two species were studied: the reduced form and the doubly disulfide cross-linked form. The proteins were totally unfolded in 6M urea-saline buffer, then refolded by tenfold dilution into benign buffer. In the refolding medium, they spontaneously recover the two-chain coiled-coil structure. Reduced beta beta refolds in at least two stages: one or more fast phases (< 0.04 s), in which an intermediate with 71% of the equilibrium ellipticity forms, followed by a slower time-resolvable phase that completes the folding. The slow phase is first order, signifying that dimerization occurs in the fast phase. The time constant of the slow phase is 2 s at 20 degrees C and requires activation parameters of delta S not equal to = -7 +/- 0.3 cal/mol.K, delta H not equal to = 15 +/- 1 kcal/mol. These results are very similar to those previously found for the reduced genetic variant alpha alpha-tropomyosin. In contrast, refolding of doubly disulfide cross-linked beta beta is complete within the dead time (< 0.04 s), whereas the singly cross-linked alpha alpha species also displays a slow phase. The opposite process, unfolding reduced beta beta from the coiled-coil state, is complete within the dead time, as in the alpha alpha variant.     

Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic alpha-helices.

The effects of 2 molal Na2SO4 at neutral pH on hydrophobic and electrostatic interactions between amphipathic alpha-helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl-LEELKKKLEELKKKLEEL-NH2) and LEE (acetyl-LEELEEELEELEEELEEL-NH2). In phosphate buffer at neutral pH, only LEK adopted a predominantly alpha-helical conformation, attributable to glu-lys+ interactions where a major contribution is evidently a hydrogen bond (Biochemistry 32: 9668-9676). Despite the presence of lys+ in the e and g' positions of the abcdefg heptad repeat, LEK exhibited mean-residue ellipticities at 222 nm ([theta]222) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na2SO4 at 25-75 degrees C, the helical content of LEK increased, with the greatest increase observed at 75 degrees C. The value of the ellipticity ratio R ([theta]222/[theta]208) of LEK in 2 molal Na2SO4 also increased, indicating a stronger interhelical association. At 50 degrees C and 75 degrees C, LEK remained predominantly alpha-helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na2SO4, however, the peptide formed alpha-helices that associated to form a coiled coil. At 50 degrees C and 75 degrees C, LEE became predominantly random coil but the remaining alpha-helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix-stabilizing and helix-destabilizing electrostatic interactions in amphipathic alpha-helices by Na2SO4.     

Increase in the stability and helical content of estrogen receptor alpha in the presence of the estrogen response element: analysis by circular dichroism spectroscopy

Ligand-dependent stabilization of the estrogen receptor (ER) is often postulated, with limited support from experimental data. We studied the thermal unfolding of recombinant ERalpha by circular dichroism (CD) spectroscopy. The T(M) of unfolding of ERalpha was 38 +/- 2.4 degrees C, and the van't Hoff enthalpy of unfolding was 31.7 +/- 3.4 kcal/mol in the absence of ligands. Addition of estradiol (E(2)) increased the T(M) to 43.6 +/- 2.3 degrees C, while addition of E(2) and an oligonucleotide harboring the estrogen response element (ERE) increased the T(M) to 47.9 +/- 1.6 degrees C. Addition of the antiestrogen 4-hydroxytamoxifen (HT) alone did not increase the T(M); however, a combination of HT and the ERE increased the T(M) to 48.9 +/- 1.0 degrees C. The ERE alone increased the T(M) to 46.1 +/- 0.9 degrees C. Addition of E(2) alone had no effect on the apparent enthalpy of unfolding; however, the ERE alone increased the apparent enthalpy from 31.7 to 36.1 kcal/mol. ERalpha samples containing the ERE also exhibited an increase in the negative ellipticity at 208 and 222 nm, relative to that of ligand-free ERalpha, suggesting a stabilization of the alpha-helix. CD data analysis further showed that the presence of the ERE caused a large increase in alpha-helical content of ERalpha in both the presence and absence of the ligands. This increase in alpha-helical content of ERalpha was not observed in the presence of a nonspecific oligonucleotide. These results show that the ERE can increase the thermal stability of ERalpha, enhance its alpha-helical content, and facilitate the cooperativity of the folding transition.     

Spectroscopic and calorimetric analyses of invasion plasmid antigen D (IpaD) from Shigella flexneri reveal the presence of two structural domains

Shigella flexneri is a facultative intracellular pathogen that causes severe gastroenteritis in humans. Invasion plasmid antigen D (IpaD) is an essential participant in Shigella invasion of intestinal cells, but no detailed structural information is available to help understand the proposed role of IpaD in invasion or its interaction with other invasion proteins. Therefore, the secondary and tertiary structure and thermal stability of IpaD as well as selected IpaD deletion mutants were investigated using Fourier transform infrared (FTIR), circular dichroism (CD), and both intrinsic and extrinsic fluorescence spectroscopies. The energetics of thermal unfolding were also evaluated by differential scanning calorimetry (DSC). Secondary-structure analysis by CD and FTIR suggests that that IpaD is primarily alpha-helical with characteristics of a intramolecular coiled coil. Thermal studies revealed that the unfolding of IpaD is a complex process consisting of two transitions centered near 59 and 80 degrees C. A comparison of the data obtained with the intact protein and selected deletion mutants indicated that the lower temperature transition is a reversible event attributable to the unfolding of a small domain located at the N terminus of IpaD. In contrast, the thermal unfolding of the proposed major and highly stable C-terminal domain was irreversible and led to protein aggregation. When the results are taken together, they strongly support the idea that IpaD has two independent folding domains.     

Kinetics and thermodynamics of the unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56

Temperature-induced reversible unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56, were studied by kinetic and thermodynamic methods, using CD spectroscopy, dynamic light scattering, and scanning calorimetry. It was found that both unfolding and refolding reactions of this protein in neutral solution in the presence of 100 mM NaCl are characterized by unusually slow kinetics, which permits detailed investigation of the mechanism of these reactions. Kinetic analyses show that the unfolding of this coiled coil represents a single-stage first-order reaction, while the refolding represents a single-stage third-order reaction. The activation enthalpy and entropy for unfolding do not depend noticeably on temperature and are both significantly greater than those for the folding reaction, which show a significant dependence on temperature. The activation heat capacity change for the unfolding reaction is close to zero, while it is quite significant for the folding reaction. The correlation between the activation and structural parameters obtained for the Lpp-56 coiled coil suggests that interhelical van der Waals interactions are disrupted in the transition state, which is nevertheless still compact, and water has not yet penetrated into the interface; the transition from the transient state to the unfolded state results in hydration of exposed apolar groups of the interface and the disruption of helices. The low propensity for the Lpp-56 strands to fold and associate is caused by the high number of charged groups at neutral pH. On one hand, these charges give rise to considerable repulsive forces destabilizing the helical conformation of the strands. On the other hand, they align the folded helices in parallel and in register so that the apolar sides face each other, and the oppositely charged groups may form salt links, which are important for the formation of the trimeric coiled coil. A decrease in pH, which eliminates the salt links, dramatically decreases the stability of Lpp-56; its structure becomes less rigid and unfolds much faster.     

Conformational analysis of a mitochondrial presequence derived from the F1-ATPase beta-subunit by CD and NMR spectroscopy.

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.     

Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle alpha-tropomyosin in an engineered chimeric protein

Tropomyosin is an alpha-helical coiled-coil protein that aligns head-to-tail along the length of the actin filament and regulates its function. The solution structure of the functionally important N terminus of a short 247-residue non-muscle tropomyosin was determined in an engineered chimeric protein, GlyTM1bZip, consisting of the first 19 residues of rat short alpha-tropomyosin and the last 18 residues of the GCN4 leucine zipper. A gene encoding GlyTM1bZip was synthesized, cloned and expressed in Escherichia coli. Triple resonance NMR spectra were analyzed with the program AutoAssign to assign its backbone resonances. Multidimensional nuclear Overhauser effect spectra, X-filtered spectra and (3)J(H(N)-H(alpha)) scalar coupling were analyzed using AutoStructure. This is the first application of this new program to determine the three-dimensional structure of a symmetric homodimer and a structure not previously reported. Residues 7-35 in GlyTM1bZip form a coiled coil, but neither end is helical. Heteronuclear (15)N-(1)H nuclear Overhauser effect data showed that the non-helical N-terminal residues are flexible. The (13)C' chemical shifts of the coiled-coil backbone carbonyl groups in GlyTM1bZip showed a previously unreported periodicity, where resonances arising from residues at the coiled-coil interface in a and d positions of the heptad repeat were displaced relatively upfield and those arising from residues in c positions were displaced relatively downfield. Heteronuclear single quantum coherence spectra, collected as a function of temperature, showed that cross-peaks arising from the alpha-helical backbone and side-chains at the coiled-coil interface broadened or shifted with T(M) values approximately 20 degrees C lower than the loss of alpha-helix measured by circular dichroism, suggesting the presence of a folding intermediate. The side-chain of Ile14, a residue essential for binding interactions, exhibited multiple conformations. The conformational flexibility of the N termini of short tropomyosins may be important for their binding specificity.     

Effects of cyclic GMP on the secondary structure of cyclic GMP dependent protein kinase and analysis of the enzyme's amino-terminal domain by far-ultraviolet circular dichroism

Far-UV circular dichroism spectra of bovine lung cyclic GMP dependent protein kinase (G-kinase) show that the enzyme contains alpha-helical and beta-pleated sheet elements. Binding of cyclic GMP changes the spectra in a way consistent with the induction of beta-sheet from random coil. Examination of the amino-terminal sequence of G-kinase indicates the presence of a strongly alpha-helical segment with several features in common with the leucine zipper motif. We propose that this sequence may be the important part of the dimerization domain of the enzyme. A synthetic peptide corresponding to amino acids 1-39 of G-kinase has a strongly alpha-helical CD spectrum, supporting the predicted secondary structure of this amino-terminal sequence. In contrast to the native enzyme, a structure reduced in alpha-helix was found when a constitutively active form of G-kinase, which lacks amino acids 1-77, was studied.