Different effects of trifluoroethanol and glycerol on the stability of tropomyosin helices and the head-to-tail complex

Tropomyosin (Tm) is a dimeric coiled-coil protein, composed of 284 amino acids (410 A), that forms linear homopolymers through head-to-tail interactions at low ionic strength. The head-to-tail complex involves the overlap of approximately nine N-terminal residues of one molecule with nine C-terminal residues of another Tm molecule. In this study, we investigate the influence of 2,2,2-trifluoroethanol (TFE) and glycerol on the stability of recombinant Tm fragments (ASTm1-142, Tm143-284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm143-284(5OHW269)) contains a 5-hydroxytryptophan (5OHW) probe at position 269 whose fluorescence is sensitive to the head-to-tail interaction and allows us to accompany titrations of Tm143-284(5OHW269) with ASTm1-142 to calculate the dissociation constant (Kd) and the interaction energy at TFE and glycerol concentrations between 0% and 15%. We observe that TFE, but not glycerol, reduces the stability of the head-to-tail complex. Thermal denaturation experiments also showed that the head-to-tail complex increases the overall conformational stability of the Tm fragments. Urea and thermal denaturation assays demonstrated that both TFE and glycerol increase the stability of the isolated N- and C-terminal fragments; however, only TFE caused a significant reduction in the cooperativity of unfolding these fragments. Our results show that these two cosolvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their opposing effects on head-to-tail complex formation.     

Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.     

Relationship between the stability of hen egg-white lysozymes mutated at sites designed to interact with alpha-helix dipoles and their secretion amounts in yeast

The positively charged lysine at the C-terminals of three long alpha-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change Delta G of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long alpha-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and Delta G of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long alpha-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.     

Specific sequences determine the stability and cooperativity of folding of the C-terminal half of tropomyosin

Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](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 urea-induced 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 beta-branched residues located at d positions.     

Relationship between the Stability of Hen Egg-White Lysozymes Mutated at Sites Designed to Interact with α-Helix Dipoles and Their Secretion Amounts in Yeast

The positively charged lysine at the C-terminals of three long α-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change ΔG of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long α-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and ΔG of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long α-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.     

A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization.

Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as alpha-amino acetylation of the N-terminus of the muscle protein. Nalpha-acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.     

The role of unstructured highly charged regions on the stability and specificity of dimerization of two-stranded alpha-helical coiled-coils: analysis of the neck-hinge region of the kinesin-like motor protein Kif3A

We investigated the folding, stability, and specificity of dimerization of the neck-hinge region (residues 356-416) of the kinesin-like protein Kif3A. We showed that the predicted coiled-coil on its own (residues 356-377) will fold autonomously in solution. We then explored the ability of oppositely charged regions to specify heterodimer formation in coiled-coils by synthesizing analogs of the neck coiled-coil region with and without various negatively and positively charged extensions to the C-terminus of the neck coiled-coil and characterizing these analogs by circular dichroism spectroscopy. The charged region alone (residues 378-416) adopted a random-coil structure and this region remained unfolded in the presence of the coiled-coil. Redox experiments demonstrated that oppositely charged regions specified the formation of a hetero-two-stranded coiled-coil. Denaturation studies with urea demonstrated a decrease in coiled-coil stability with the addition of negatively charged residues in the homostranded coiled-coil; conversely, the addition of the positively charged region (residues 403-416) of Kif3A C-terminally to the neck coiled-coil did not affect coiled-coil stability. Overall, our results suggest that electrostatic attractions drive the specificity of heterodimerization of the coiled-coil, not the removal of positive or negative charge-charge repulsions, while maintaining the stability of the heterodimer compared to that of the stablest homodimer.     

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

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.     

Terminal ion pairs stabilize the second beta-hairpin of the B1 domain of protein G

The effects of terminal ion pairs on the stability of a beta-hairpin peptide corresponding to the C-terminal residues of the B1 domain of protein G were determined using thermal unfolding as monitored by nuclear magnetic resonance and circular dichroism spectroscopy. Molecular dynamics (MD) simulations were also performed to examine the effect of ion pairs on the structures. Eight peptides were studied including the wild type (G41) and the N-terminal modified sequences that had the first residue deleted (E42), replaced with a Lys (K41), or extended by an additional Gly (G40). Acetylated variants were made to examine the effect of removing the positive N-terminal charge on beta-hairpin stability. The rank in stability determined experimentally is K41 > E42 approximately G41 approximately G40 > Ac-K41 > Ac-E42 approximately Ac-G41 > Ac-G40. The Tm of the K41 peptide is 12 degrees C higher than G41, while the Tm values for the acetylated peptides are less than their unacetylated forms by more than 15 degrees C. NOE cross-peaks between side-chain methylene groups at the N- and C-termini and larger CalphaH shifts compared to random values are seen for K41. The addition of 20% methanol increases the stability in K41 and G41. The MD studies complement these results by showing that the charged N-terminus is important to stability. The type of ion pair observed varies with peptide, and when formed the simulations show that the ion pair can prevent fraying of the beta-strands through electrostatic and hydrophobic contacts. Therefore, introducing favorable electrostatic interactions at the N- and C-termini can substantially enhance beta-hairpin stability and help define the structure.     

Conserved noncanonical residue Gly-126 confers instability to the middle part of the tropomyosin molecule

Tropomyosin (Tm) is a two-stranded α-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle α-Tm either with an Ala residue, which should stabilize the Tm α-helix, or with an Arg residue, which is expected to stabilize both α-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations confer cooperativity to this part of the Tm molecule. The size of the flexible middle part of Tm is estimated to be 60-70 amino acid residues, about a quarter of the Tm molecule. Thus, our results show that flexibility is unevenly distributed in the Tm molecule and achieves the highest extent in its middle part. We conclude that the highly conserved Gly-126, acting in concert with the previously identified non-canonical Asp-137, destabilizes the middle part of Tm, resulting in a more flexible region that is important for Tm function.     

Stability and specificity of heterodimer formation for the coiled-coil neck regions of the motor proteins Kif3A and Kif3B: the role of unstructured oppositely charged regions.

We investigated the folding, stability, and specificity of dimerization of the neck regions of the kinesin-like proteins Kif3A (residues 356-416) and Kif3B (residues 351-411). We showed that the complementary charged regions found in the hinge regions (which directly follow the neck regions) of these proteins do not adopt any secondary structure in solution. We then explored the ability of the complementary charged regions to specify heterodimer formation for the neck region coiled-coils found in Kif3A and Kif3B. Redox experiments demonstrated that oppositely charged regions specified the formation of a heterodimeric coiled-coil. Denaturation studies with urea demonstrated that the negatively charged region of Kif3A dramatically destabilized its neck coiled-coil (urea1/2 value of 3.9 m compared with 6.7 m for the coiled-coil alone). By comparison, the placement of a positively charged region C-terminal to the neck coiled-coil of Kif3B had little effect on stability (urea1/2 value of 8.2 m compared with 8.8 m for the coiled-coil alone). The pairing of complementary charged regions leads to specific heterodimer formation where the stability of the heterodimeric neck coiled-coil with charged regions had similar stability (urea1/2 value of 7.8 m) to the most stable homodimer (Kif3B) with charged regions (urea1/2 value of 8.0 m) and dramatically more stable than the Kif3A homodimer with charged regions (urea1/2, value of 3.9 m). The heterodimeric coiled-coil with charged extensions has essentially the same stability as the heterodimeric coiled-coil on its own (urea1/2 values of 7.8 and 8.1 m, respectively) suggesting that specificity of heterodimerization is driven by non-specific attraction of the oppositely unstructured charged regions without affecting stability of the heterodimeric coiled-coil.     

The structure of the carboxyl terminus of striated alpha-tropomyosin in solution reveals an unusual parallel arrangement of interacting alpha-helices

Coiled coils are well-known as oligomerization domains, but they are also important sites of protein-protein interactions. We determined the NMR solution structure and backbone (15)N relaxation rates of a disulfide cross-linked, two-chain, 37-residue polypeptide containing the 34 C-terminal residues of striated muscle alpha-tropomyosin, TM9a(251-284). The peptide binds to the N-terminal region of TM and to the tropomyosin-binding domain of the regulatory protein, troponin T. Comparison of the NMR solution structure of TM9a(251-284) with the X-ray structure of a related peptide [Li, Y., Mui, S., Brown, J. H., Strand, J., Reshetnikova, L., Tobacman, L. S., and Cohen, C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 7378-7383] reveals significant differences. In solution, residues 253-269 (like most of the tropomyosin molecule) form a canonical coiled coil. Residues 270-279, however, are parallel, linear helices, novel for tropomyosin. The packing between the parallel helices results from unusual interface residues that are atypical for coiled coils. Y267 has poor packing at the coiled-coil interface and a lower R(2) relaxation rate than neighboring residues, suggesting there is conformational flexibility around this residue. The last five residues are nonhelical and flexible. The exposed surface presented by the parallel helices, and the flexibility around Y267 and the ends, may facilitate binding to troponin T and formation of complexes with the N-terminus of tropomyosin and actin. We propose that unusual packing and flexibility are general features of coiled-coil domains in proteins that are involved in intermolecular interactions.     

Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.     

Clustering of large hydrophobes in the hydrophobic core of two-stranded alpha-helical coiled-coils controls protein folding and stability

The de novo design and biophysical characterization of two 60-residue peptides that dimerize to fold as parallel coiled-coils with different hydrophobic core clustering is described. Our goal was to investigate whether designing coiled-coils with identical hydrophobicity but with different hydrophobic clustering of non-polar core residues (each contained 6 Leu, 3 Ile, and 7 Ala residues in the hydrophobic core) would affect helical content and protein stability. The disulfide-bridged P3 and P2 differed dramatically in alpha-helical structure in benign conditions. P3 with three hydrophobic clusters was 98% alpha-helical, whereas P2 was only 65% alpha-helical. The stability profiles of these two analogs were compared, and the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence by circular dichroism spectroscopy and confirmed by differential scanning calorimetry. The results showed that P3 assembled into a stable alpha-helical two-stranded coiled-coil and exhibited a native protein-like cooperative two-state transition in thermal melting, chemical denaturation, and calorimetry experiments. Although both peptides have identical inherent hydrophobicity (the hydrophobic burial of identical non-polar residues in equivalent heptad coiled-coil positions), we found that the context dependence of an additional hydrophobic cluster dramatically increased stability of P3 (Delta Tm approximately equal to 18 degrees C and Delta[urea](1/2) approximately equal to 1.5 M) as compared with P2. These results suggested that hydrophobic clustering significantly stabilized the coiled-coil structure and may explain how long fibrous proteins like tropomyosin maintain chain integrity while accommodating polar or charged residues in regions of the protein hydrophobic core.     

Real-time monitoring of the interactions of two-stranded de novo designed coiled-coils: effect of chain length on the kinetic and thermodynamic constants of binding

We have de novo designed a heterodimeric coiled-coil formed by two peptides as a capture/delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coil (KVSALKE heptad sequence) and E coil (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 microM (E3/K3). From these K(d) values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the coiled-coils. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.     

Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a high-affinity nitrate transporter

The transport of nitrate into prokaryotic and eukaryotic cells, of considerable interest to agriculture, ecology, and human health, is carried out by members of a distinct cluster of proteins within the major facilitator superfamily. To obtain structure/function information on this important class of nitrate permeases, a collection of chemically induced mutations in the nrtA gene encoding a 12-transmembrane domain, high-affinity nitrate transporter from the eukaryote Aspergillus nidulans was isolated and characterized. This mutational analysis, coupled with protein alignments, demonstrates the utility of the approach to predicting peptide motifs and individual residues important for the movement of nitrate across the membrane. These include the highly conserved nitrate signature motif (residues 166-173) in Tm 5, the conserved charged residues Arg87 (Tm 2) and Arg368 (Tm 8), as well as the aromatic residue Phe47 (Tm 1), all within transmembrane helices. No mutations were observed in the large central loop (Lp 6/7) between Tm 6 and Tm 7. Finally, the study of a strain with a conversion of Trp481 (Tm 12) to a stop codon suggests that all 12 transmembrane domains and/or the C-terminal tail are required for membrane insertion and/or stability of NrtA.     

Folding and stability of a coiled-coil investigated using chemical and physical denaturing agents: comparative analysis of polymerized and non-polymerized forms of alpha-tropomyosin

alpha-Tropomyosin (Tm) is a two-stranded alpha-helical coiled-coil protein, which participates in the regulation of muscle contraction. Unlike Tm purified from vertebrate muscle, recombinant Tm expressed in Escherichia coli is not acetylated at the N-terminal residue and loses the capacity to undergo head-to-tail polymerization, to bind actin and to inhibit actomyosin ATPase activity. These functions are restored by fusion of an N-terminal Ala-Ser (AS) dipeptide tail to recombinant Tm. Here, we have employed chemical (guanidine hydrochloride and urea) and physical (elevated hydrostatic pressures and low temperatures) denaturing agents to compare the structural stabilities of polymeric alanine-serine-tropomyosin (ASTm, containing the AS dipeptide) and dimeric "non-fusion" Tm (nfTm, i.e., not containing the AS dipeptide). Binding of the hydrophobic fluorescent dye bis-ANS, circular dichroism and size-exclusion chromatography were used to monitor the stabilities and state of association of both proteins under different solution conditions. Bis-ANS binding was markedly decreased at low concentrations (<1M) of GdnHCl or urea, whereas the secondary structures of both ASTm and nfTm were essentially unaffected in the same range of denaturant concentrations. These results suggest local unfolding of bis-ANS binding domains prior to global unfolding of Tm. In contrast, increased bis-ANS binding was observed when Tm was submitted to high pressures or to low temperatures, implying increased exposure of hydrophobic domains in the protein. Taken together, the different sensitivities of ASTm and nfTm to different denaturing agents support the notion that, at close to physiological conditions, head-to-tail interactions in polymerized ASTm are predominantly stabilized by electrostatic interactions between adjacent Tm dimers, whereas non-polar interactions appear to play a major role in the stability of the coiled-coil structure of individual Tm dimers.     

Acid-induced polymerization of the group 5 mite allergen from Dermatophagoides pteronyssinus.

House dust mites are the most important source of indoor allergens and cause allergic diseases. Our studies here suggest that the group 5 allergen from Dermatophagoides pteronyssinus (Der p 5) is monomeric at neutral pH, but forms filaments at low pH. Circular dichroism measurements show Der p 5 is a helical protein, and the protein sequence reveals Der p 5 contains coiled-coil helices. The acid-induced filament assembly could be explained in part by the high content of charged residues (40%) in the coiled-coil structure. Interestingly, some of the known Dermatophagoides allergens also contain a heptad repeat, which could potentially form coiled coils. Therefore, coiled-coil helices may be one of the common structural motifs of mite allergens that contribute to their allergenicity.     

Pitch diversity in α-helical coiled coils

Two complementary methods for measuring local pitch based on heptad position in α-helical coiled coils are described and applied to six crystal structures. The results reveal a diversity of pitch values: two-stranded coiled coils appear to have pitch values near 150 Å the values for three- and four-stranded coiled coils range closer to 200 Å. The methods also provide a rapid and sensitive gauge of local coiled-coil conformation. Polar or charged residues in the apolar interface between coiled-coil helices markedly affect local pitch values, suggesting a connection between pitch uniformity and coiled-coil stability. Moreover, the identification of a skip residue (heptad frame shift) in the hemaglutinin glycoprotein of influenza virus (HA) allows interpretation of local pitch changes. These results on relatively short coiled-coil structures have relevance for the much longer fibrous proteins (many of which have skip residues) whose detailed structures are not yet established. We also show that local pitch values from molecular dynamics predictions of the GCN4 leucine zipper are in striking agreement with the high-resolution crystal structure—a result not readily discerned by direct comparison of atomic coordinates. Taken together, these methods reveal specific aspects of coiled-coil structure which may escape detection by global analyses of pitch. © 1993 Wiley-Liss, Inc.