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.     

pH-induced folding of an apoptotic coiled coil

Par-4 is a 38-kD protein pivotal to the apoptotic pathways of various cell types, most notably prostate cells and neurons, where it has been linked to prostate cancer and various neurodegenerative disorders including Alzheimer's and Huntington's diseases and HIV encephalitis. The C-terminal region of Par-4 is responsible for homodimerization and the ability of Par-4 to interact with proposed effector molecules. In this study, we show that the C-terminal 47 residues of Par-4 are natively unfolded at physiological pH and temperature. Evidence is rapidly accumulating that natively unfolded proteins play an important role in various cellular functions and signaling pathways, and that folding can often be induced on complexation with effector molecules or alteration of environment. Here we use primarily CD studies to show that changes in the environment, particularly pH and temperature, can induce the Par-4 C terminus to form a self-associated coiled coil.     

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.     

Beta beta homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation.

Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 3-4/1). In some extant studies, no beta beta molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of beta beta molecules cross-linked at both C36 and C190 (beta = beta). In such cross-linked RSTm, 3-8% beta = beta is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes approximately 4-10% beta beta when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed beta beta species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55 degrees C or by incubating at 45 degrees C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of beta beta increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138).     

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein.

Two equilibrium intermediates have previously been observed in the urea denaturation of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded beta/alpha barrel protein. In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105-214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth beta-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of betaalphabeta supersecondary structural elements. One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four betaalpha units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in alphaTS requires the participation of hydrophobic residues in multiple beta-strands that span the entire sequence.     

Unique stabilizing interactions identified in the two-stranded alpha-helical coiled-coil: crystal structure of a cortexillin I/GCN4 hybrid coiled-coil peptide

We determined the 1.17 A resolution X-ray crystal structure of a hybrid peptide based on sequences from coiled-coil regions of the proteins GCN4 and cortexillin I. The peptide forms a parallel homodimeric coiled-coil, with C(alpha) backbone geometry similar to GCN4 (rmsd value 0.71 A). Three stabilizing interactions have been identified: a unique hydrogen bonding-electrostatic network not previously observed in coiled-coils, and two other hydrophobic interactions involving leucine residues at positions e and g from both g-a' and d-e' interchain interactions with the hydrophobic core. This is also the first report of the quantitative significance of these interactions. The GCN4/cortexillin hybrid surprisingly has two interchain Glu-Lys' ion pairs that form a hydrogen bonding network with the Asn residues in the core. This network, which was not observed for the reversed Lys-Glu' pair in GCN4, increases the combined stability contribution of each Glu-Lys' salt bridge across the central Asn15-Asn15' core to approximately 0.7 kcal/mole, compared to approximately 0.4 kcal mole(-1) from a Glu-Lys' salt bridge on its own. In addition to electrostatic and hydrogen bonding stabilization of the coiled-coil, individual leucine residues at positions e and g in the hybrid peptide also contribute to stability by 0.7 kcal/mole relative to alanine. These interactions are of critical importance to understanding the stability requirements for coiled-coil folding and in modulating the stability of de novo designed macromolecules containing this motif.     

Deciphering the role of the electrostatic interactions in the alpha-tropomyosin head-to-tail complex

Skeletal alpha-tropomyosin (Tm) is a dimeric coiled-coil protein that forms linear assemblies under low ionic strength conditions in vitro through head-to-tail interactions. A previously published NMR structure of the Tm head-to-tail complex revealed that it is formed by the insertion of the N-terminal coiled-coil of one molecule into a cleft formed by the separation of the helices at the C-terminus of a second molecule. To evaluate the contribution of charged residues to complex stability, we employed single and double-mutant Tm fragments in which specific charged residues were changed to alanine in head-to-tail binding assays, and the effects of the mutations were analyzed by thermodynamic double-mutant cycles and protein-protein docking. The results show that residues K5, K7, and D280 are essential to the stability of the complex. Though D2, K6, D275, and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to head-to-tail complex stability by modulating the stability of the helices at the Tm termini.     

Folding of a three-stranded coiled coil

Coiled coils consist of two or more amphipathic a-helices wrapped around each other to form a superhelical structure stabilized at the interhelical interface by hydrophobic residues spaced in a repeating 3-4 sequence pattern. Dimeric coiled coils have been shown to often form in a single step reaction in which association and folding of peptide chains are tightly coupled. Here, we ask whether such a simple folding mechanism may also apply to the formation of a three-stranded coiled coil. The designed 29-residue peptide LZ16A was shown previously to be in a concentration-dependent equilibrium between unfolded monomer (M), folded dimer (D), and folded trimer (T). We show by time-resolved fluorescence change experiments that folding of LZ16A to D and T can be described by 2M (k1)<==>(k(-1)) D and M + D (k2)<==>(k(-2)) T. The following rate constants were determined (25 degrees C, pH 7): k1 = 7.8 x 10(4) M(-1) s(-1), k(-1) = 0.015 s(-1), k2 = 6.5 x 10(5) M(-1) s(-1), and k(-2) = 1.1 s(-1). In a separate experiment, equilibrium binding constants were determined from the change with concentration of the far-ultraviolet circular dichroism spectrum of LZ16A and were in good agreement with the kinetic rate constants according to K(D) = k1/2k(-1) and K(T) = k2/k(-2). Furthermore, pulsed hydrogen-exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The results are compatible with a two-step reaction in which a subpopulation of association competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.     

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.     

Toward an improved structural model of the frog-skin antimicrobial peptide esculentin-1b(1-18)

Antimicrobial peptides (AMPs) are found in various classes of organisms as part of the innate immune system. Despite high sequence variability, they share common features such as net positive charge and an amphipathic fold when interacting with biologic membranes. Esculentin-1b is a 46-mer frog-skin peptide, which shows an outstanding antimicrobial activity. Experimental studies revealed that the N-terminal fragment encompassing the first 18 residues, Esc(1-18), is responsible for the antimicrobial activity of the whole peptide, with a negligible toxicity toward eukaryotic cells, thus representing an excellent candidate for future pharmaceutical applications. Similarly to most of the known AMPs, Esc(1-18) is expected to act by destroying/permeating the bacterial plasma-membrane but, to date, its 3D structure and the detailed mode of action remains unexplored. Before an in-depth investigation on peptide/membranes interactions could be undertaken, it is necessary to characterize peptide's folding propensity in solution, to understand what is intrinsically due to the peptide sequence, and what is actually driven by the membrane interaction. Circular dichroism and nuclear magnetic resonance spectroscopy were used to determine the structure adopted by the peptide, moving from water to increasing amounts of trifluoroethanol. The results showed that Esc(1-18) has a clear tendency to fold in a helical conformation as hydrophobicity of the environment increases, revealing an intriguing amphipathic structure. The helical folding is adopted only by the N-terminal portion of the peptide, while the rest is unstructured. The presence of a hydrophobic cluster of residues in the C-terminal portion suggests its possible membrane-anchoring role.     

Soft metal ions, Cd(II) and Hg(II), induce triple-stranded alpha-helical assembly and folding of a de novo designed peptide in their trigonal geometries

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded alpha-helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple-stranded alpha-helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 +/- 0.8 microM for Cd(II). The triple-stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl- ions. The 113Cd NMR resonance was connected with the betaH protons of the cysteine residue by 1H-113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg(II) also induced the assembly of the peptide into a triple-stranded alpha-helical bundle below the Hg(II)/peptide ratio of 1/3. With excess Hg(II), however, the alpha-helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.     

The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli.

The urea-induced equilibrium unfolding of the alpha subunit of tryptophan synthase (alphaTS), a single domain alpha/beta barrel protein, displays a stable intermediate at approximately 3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784-792). The same experiment, monitored by one-dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab-Rincón G et al., 1993, Biochemistry 32:13,981-13,990). To further test and quantify the implied four-state model, N <--> I1 <--> I2 <--> U, the urea-induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far-UV CD. All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method. Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol(-1), respectively. I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers approximately 6% of the native CD signal. This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone. I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N. The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of alphaTS.     

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.     

Conformational studies of alanine-rich peptide using CD and FTIR spectroscopy.

The circular dichroism (CD) and Fourier transform infrared (FTIR) methods were applied to the conformational studies of alanine-rich peptide Ac-K-[A]11-KGGY-NH2 (where K is lysine, A is alanine, G is glycine and Y is tyrozyne) in water, methanol (MeOH) and trifluoroethanol (TFE). The analysis of CD-spectra of the peptide in water at different concentrations revealed that the secondary structure content depends on the peptide concentration and pH of the solution. The increase of the peptide concentration causes a decrease of alpha-helix content and, simultaneously, an increase of beta-sheet structure, while the unordered structure is the predominant one. Additional elements are discovered in MeOH and TFE but alpha-helix and beta-turns predominate. Moreover, in these solutions the percentage content of the secondary structure does not depend on the temperature. FTIR measurements, carried out at higher peptide concentration (about one order of magnitude) than these CD measurements mentioned above, revealed that in water solution the solid state beta-sheet, and aggregated structures, dominate. However, in TFE the most abundant are alpha-helix and beta-turns structures. The thioflavine T assay showed the tendency of the studied peptide for aggregate.     

Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization

The folding kinetics of human common-type acylphosphatase (cAcP) from its urea- and TFE-denatured states have been determined by stopped-flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53-Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly a-helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53-Gly69 of cAcP indicate that only the native-like trans isomer of the Gly-Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an approximately 1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53-Pro54 peptide bond is stabilized in TFE by the formation of a nonnative-like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.     

Synthesis and conformational characterization of peptides related to the neck domain of a fungal kinesin.

The Y362K mutation in the neck domain of conventional kinesin from Neurospora crassa provokes a significant reduction of the rate of movement along microtubules. Since the alpha-helical coiled-coil structure of the neck region is implicated in the mechanism of the processive movement of kinesins, a series of peptides related to the heptad region 338-379 of the wild-type and the variant fungal kinesinswere synthesized as monomers and as N-terminal disulfide dimers, crosslinked to favour self-association into coiled-coil structures entropically. A comparison of the dichroic properties of the peptides and the effects of trifluoroethanol and peptide concentration clearly confirmed the strong implication of the single point mutation in destabilizing the intrinsic propensity of the peptides to fold into the supercoiled conformation. That there is a correlation between the stability of the coiled-coil and rate of movement of the kinesin is confirmed.