A desiccated dual-species subaerial biofilm reprograms its metabolism and affects water dynamics in limestone.

Understanding the impact of sessile communities on underlying materials is of paramount importance in stone conservation. Up until now, the critical role of subaerial biofilms (SABs) whether they are protective or deteriorative remains unclear, especially under desiccation. The interest in desiccated SABs is raised by the prediction of an increase in drought events in the next decades that will affect the Mediterranean regions' rich stone heritage as never before. Thus, the main goal of this research is to study the effects of desiccation on both the biofilms' eco-physiology and its impacts on the lithic substrate. To this end, we used a dual-species model system composed of a phototroph and a chemotroph to simulate biofilm behavior on stone heritage. We found that drought altered the phototroph-chemotroph balance and enriched the biofilm matrix with proteins and DNA. Desiccated SABs underwent a shift in metabolism to fermentation and a decrease in oxidative stress. Additionally, desiccated SABs changed the water-related dynamics (adsorption, evaporation, and wetting properties) in limestone. Water absorption experiments showed that desiccated SABs protected the stone from rapid water uptake, while a thermographic survey indicated a delay in water evaporation. Spilling-drop tests revealed a change in the wettability of the stone-SAB interface, which affected the water transport properties of the stone. Finally, desiccated SABs reduced stone swelling in the presence of water vapor. The biodeteriorative and bioprotective implications of desiccated SABs on the stone were ultimately assessed.

Monomeric and dimeric states exhibited by the kinesin-related motor protein KIF1A.

KIF1A, a kinesin-related motor protein that transports pre-synaptic vesicles in neurons, was originally presumed to translocate along microtubules (MT) as a monomer. Protein structure predictions from its amino acid sequence failed to identify the long coiled-coil domains typical of kinesins, which led researchers to believe it does not oligomerize into the canonical kinesin dimer. However, mounting evidence using recombinant chimeric protein indicates that KIF1A, like conventional kinesin, requires dimerization for fast, unidirectional processive movement along MTs. Because these studies are somewhat indirect, we wished to test the oligomerization state of native KIF1A, and to compare that to full-length recombinant protein. We have performed hydrodynamic analyses to determine the molecular weights of the respective complexes. Our results indicate that most native KIF1A is soluble and indeed monomeric, but recombinant KIF1A is a dimer. MT-binding studies also showed that native KIF1A did not bind to MTs in either the presence of AMP-PNP, apyrase, or adenosine triphosphate (ATP), but recombinant KIF1A bound to MTs most stably in the presence of ATP, indicating very different motor functional states. To further characterize KIF1A's dimerization potential, we prepared peptides corresponding to the neck domains of MmKIF1A and CeUnc104, and by circular dichroism spectroscopy compared these peptides for their ability to form coiled-coils. Interestingly, both MmKIF1A and CeUnc104 neck peptides formed homodimeric coiled-coils, with the MmKIF1A neck coiled-coil exhibiting the greater stability. Collectively, from our data and from previous studies, we predict that native KIF1A can exist as both an inactive monomer and an active homodimer formed in part through its neck coiled-coil domain.

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.

Mapping the interacting regions between troponins T and C. Binding of TnT and TnI peptides to TnC and NMR mapping of the TnT-binding site on TnC.

Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex. The interactions among the components of the troponin complex (troponins T, C, and I) are essential to understanding the regulation of muscle contraction. While the structure of TnC is well known, and a model for the binary TnC.TnI complex has been recently published (Tung, C.-S., Wall, M. E., Gallagher, S. C., and Trewhella, J. (2000) Protein Sci. 9, 1312-1326), very little is known about TnT. Using non-denaturing gels and NMR spectroscopy, we have analyzed the interactions between TnC and five peptides from TnT as well as how three TnI peptides affect these interactions. Rabbit fast skeletal muscle peptide TnT-(160-193) binds to TnC with a dissociation constant of 30 +/- 6 microm. This binding still occurs in the presence of TnI-(1-40) but is prevented by the presence of TnI-(56-115) or TnI-(96-139), both containing the primary inhibitory region of TnI. TnT-(228-260) also binds TnC. The binding site for TnT-(160-193) is located on the C-terminal domain of TnC and was mapped to the surface of TnC using NMR chemical shift mapping techniques. In the context of the model for the TnC.TnI complex, we discuss the interactions between TnT and the other troponin subunits.

The role of the carboxyl terminal alpha-helical coiled-coil domain in osmosensing by transporter ProP of Escherichia coli.

Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.

Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position "d".

We describe the de novo design and biophysical characterization of a model coiled-coil protein in which we have systematically substituted 20 different amino acid residues in the central "d" position. The model protein consists of two identical 38 residue polypeptide chains covalently linked at their N termini via a disulfide bridge. The hydrophobic core contained Val and Ile residues at positions "a" and Leu residues at positions "d". This core allowed for the formation of both two-stranded and three-stranded coiled-coils in benign buffer, depending on the substitution at position "d". The structure of each analog was analyzed by CD spectroscopy and their relative stability determined by chemical denaturation using GdnHCI (all analogs denatured from the two-stranded state). The oligomeric state(s) was determined by high-performance size-exclusion chromatography and sedimentation equilibrium analysis in benign medium. Our results showed a thermodynamic stability order (in order of decreasing stability) of: Leu, Met, Ile, Tyr, Phe, Val, Gln, Ala, Trp, Asn, His, Thr, Lys, Ser, Asp, Glu, Arg, Orn, and Gly. The Pro analog prevented coiled-coil formation. The overall stability range was 7.4 kcal/mol from the lowest to the highest analog, indicating the importance of the hydrophobic core and the dramatic effect a single substitution in the core can have upon the stability of the protein fold. In general, the side-chain contribution to the level of stability correlated with side-chain hydrophobicity. Molecular modelling studies, however, showed that packing effects could explain deviations from a direct correlation. In regards to oligomerization state, eight analogs demonstrated the ability to populate exclusively one oligomerization state in benign buffer (0.1 M KCl, 0.05 M K(2)PO(4)(pH 7)). Ile and Val (the beta-branched residues) induced the three-stranded oligomerization state, whereas Tyr, Lys, Arg, Orn, Glu and Asp induced the two-stranded state. Asn, Gln, Ser, Ala, Gly, Phe, Leu, Met and Trp analogs were indiscriminate and populated two-stranded and three-stranded states. Comparison of these results with similar substitutions in position "a" highlights the positional effects of individual residues in defining the stability and numbers of polypeptide chains occurring in a coiled-coil structure. Overall, these results in conjunction with other work now generate a relative thermodynamic stability scale for 19 naturally occurring amino acid residues in either an "a" or "d" position of a two-stranded coiled-coil. Thus, these results will aid in the de novo design of new coiled-coil structures, a better understanding of their structure/function relationships and the design of algorithms to predict the presence of coiled-coils within native protein sequences.

The role of position a in determining the stability and oligomerization state of alpha-helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins.

We describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled-coil protein in determining coiled-coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three-stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys-Gly-Gly linker, unfolding occurred from the same two-stranded monomeric oligomerization state for all of the analogs. Denaturation from a two-stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled-coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed-phase high-performance liquid chromatography and found to correlate very highly (R = 0.95) with coiled-coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high-performance size-exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 microM under benign buffer conditions. The Leu-, Tyr-, Gln-, and His-substituted analogs were found to be exclusively three-stranded trimers, while the Asn-, Lys-, Orn-, Arg-, and Trp-substituted analogs formed exclusively two-stranded monomers. Modeling results for the Leu-substituted analog showed that a three-stranded oligomerization state is preferred due to increased side-chain burial, while a two-stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three-stranded state.

Defining the region of troponin-I that binds to troponin-C.

The kinetics and energetics of the binding of three troponin-I peptides, corresponding to regions 96-131 (TnI96-131), 96-139 (TnI96-139), and 96-148 (TnI96-148), to skeletal chicken troponin-C were investigated using multinuclear, multidimensional NMR spectroscopy. The kinetic off-rate and dissociation constants for TnI96-131 (400 s-1, 32 microM), TnI96-139 (65 s-1, <1 microM), and TnI96-148 (45 s-1, <1 microM) binding to TnC were determined from simulation and analysis of the behavior of 1H,15N-heteronuclear single quantum correlation NMR spectra taken during titrations of TnC with these peptides. Two-dimensional 15N-edited TOCSY and NOESY spectroscopy were used to identify 11 C-terminal residues from the 15N-labeled TnI96-148 that were unperturbed by TnC binding. TnI96-139 labeled with 13C at four positions (Leu102, Leu111, Met 121, and Met134) was complexed with TnC and revealed single bound species for Leu102 and Leu111 but multiple bound species for Met121 and Met134. These results indicate that residues 97-136 (and 96 or 137) of TnI are involved in binding to the two domains of troponin-C under calcium saturating conditions, and that the interaction with the regulatory domain is complex. Implications of these results in the context of various models of muscle regulation are discussed.

Multiple distinct coiled-coils are involved in dynamin self-assembly.

Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes. Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission. In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers. In this study we sought to define self-assembly regions in dynamin. Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S. Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site. Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer. Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants. Self-association was also observed between different dynamin isoforms. Taken altogether, our results reveal two distinct coiled-coil-containing assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition. In addition, our data strongly suggests a parallel model for dynamin subunit self-association.

De novo design of a model peptide sequence to examine the effects of single amino acid substitutions in the hydrophobic core on both stability and oligomerization state of coiled-coils.

A model peptide sequence was de novo designed to investigate the hydrophobicity, stability and oligomerization state resulting from single amino acid substitutions in the hydrophobic core a position of the central heptad of a five heptad coiled-coil. This involved selecting a hydrophobic core consisting of Val and Leu at a and d positions, respectively, known to form both two- and three-stranded coiled-coils. In addition, the sequence provided the correct overall coiled-coil stability and maximized the hydrophobicity surrounding the substitution site by having Leu in the hydrophobic core above and below the site of substitution. To control oligomerization state we exploited differential placement of an interhelical disulfide-bridge, which was placed in the coiled-coil hydrophobic core at the C-terminal d position, or alternatively outside of the core at the N-terminal via a Cys-Gly-Gly linker. We found that the Cys-Gly-Gly linker allowed assessment of both relative stability and oligomerization state after extensive biophysical characterization of the models by circular dichroism, sedimentation equilibrium, sedimentation velocity and finally high-performance size-exclusion chromatography (HPSEC) under both benign and denaturing conditions. The Cys-Gly-Gly linker was found to be unique in allowing the inherent two- or three-stranded oligomerization state to be observed in benign medium, while also allowing the stability to be determined by concentration independent chemical denaturation of a two-stranded coiled-coil. This entails a two-state transition from a folded disulfide-bridged two-stranded coiled-coil (monomeric state) to the unfolded monomer, even for analogs where the coiled-coil is a trimer of disulfide-bridged peptides in benign medium. We also developed novel HPSEC methodology for monitoring the chemical denaturation of a folded monomeric protein in fast exchange with the corresponding unfolded protein, which elutes as a single peak throughout the denaturation process.

Structural cassette mutagenesis in a de novo designed protein: proof of a novel concept for examining protein folding and stability.

The solution to the protein folding problem lies in defining the relative energetic contributions of short-range and long-range interactions. In other words, the tendency of a stretch of amino acids to adopt a final secondary structural fold is context dependent. Our approach to this problem is to address whether an amino acid sequence, a "cassette," with a defined secondary structure in the three-dimensional structure of a native protein, can adopt a different conformation when placed into a different protein environment. Thus, we designed de novo a disulfide-bridged two-stranded alpha-helical parallel coiled coil, where each polypeptide chain consisted of 39 residues, as a "cassette holder." The 11-residue cassette would be inserted into the center of each polypeptide chain between the two nucleating alpha-helices to replace the control sequence. This Structural Cassette Mutagenesis model permits the analysis of short-range interactions within the inserted cassette as well as long-range interactions between the nucleating helices and the cassette region. The cassette holder, with a control sequence as the cassette, had a GdnHCl transition midpoint during denaturation of 5.6M. To demonstrate the feasibility of our model, an 11-residue beta-strand cassette from an immunoglobulin fold was inserted. The cassette was fully induced into the alpha-helical conformation with a [GdnHCl]1/2 value of 3.2M. To demonstrate the importance of short-range interactions (beta-sheet/alpha-helical propensities of amino acid side chains) in modulating structure and stability, a series of 1-5 threonine residues (highest beta-sheet propensity) were substituted into the solvent-exposed portions of the cassette in the alpha-helical conformation. Each successive substitution systematically decreased the stability of the coiled coil with peptide T4b (4 Thr residues) having a [GdnHCl]1/2 value of 2.2M. The single substitution of Ile in the hydrophobic core of the cassette with Ala or Thr had the most dramatic effect on protein stability (peptide 120T, [GdnHCl]1/2 value of 1.4M). Though these substitutions were able to modulate stability, they were not able to disrupt the alpha-helical conformation of the cassette, showing the importance of the nucleating alpha-helices on either side of the cassette in controlling conformation of the cassette. We have demonstrated the feasibility of our model protein to accept a beta-strand cassette. The effect of cassettes containing other beta-strands, beta-turns, loops, regions of undefined structure, and helical segments on conformation and stability of our model protein will also be determined.

A strategy for rational design of fully synthetic glycopeptide conjugate vaccines.

The present study describes a strategy to rationally design fully synthetic glycopeptide conjugate vaccines. Glycopeptide immunogens were constructed by coupling synthetic oligosaccharides comprising repeating units of synthetic 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate (sPRP) to synthetic peptides containing potent T-helper cell determinants and B-cell epitopes of the Haemophilus influenzae type b (Hib) outer membrane proteins (OMPs) P1, P2, and P6. Rabbit immunogenicity studies revealed that some of these fully synthetic glycoconjugates were capable of eliciting high titers of both anti-PRP and anti-OMP immunoglobulin G antibodies. In addition, we systematically investigated the factors which could influence their immunogenicity. We observed that the magnitude of the anti-PRP antibody response markedly depended on the relative spatial orientation of sPRP and T-cell epitopes, the anti-PRP antibody response was enhanced when a multiple antigenic peptide was used as a carrier, the anti-PRP antibody response was optimal for three PRP repeating units, and lipidation of peptide-PRP conjugates had a minimal effect on the magnitude of the anti-PRP antibody response. The results of this study clearly demonstrate that coupling a carbohydrate hapten to a peptide can provide T-cell help and convert it into a T-cell-dependent antigen. The antisera raised against these conjugates were also found to be protective against Hib infection in the infant rat model of bacteremia.

Interaction of the second binding region of troponin I with the regulatory domain of skeletal muscle troponin C as determined by NMR spectroscopy.

Two dimensional 1H,15N-heteronuclear single quantum correlation NMR was used to monitor the resonance frequency changes of the backbone amide groups belonging to the 15N-labeled regulatory domain of calcium saturated troponin C (N-TnC) upon addition of synthetic skeletal N-acetyl-troponin I 115-131-amide peptide (TnI115-131). Utilizing the change in amide chemical shifts, the dissociation constant for 1:1 binding of TnI115-131 to N-TnC in low salt and 100 mM KCl samples was determined to be 28 +/- 4 and 24 +/- 4 microM, respectively. The off rate of TnI115-131 was determined to be 300 s-1 from observed N-TnC backbone amide 1H,15N-heteronuclear single quantum correlation cross-peak line widths, which is on the order of the calcium off rates (Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry 34, 8330-8340), and agrees with kinetic expectations for biological regulation of muscle contraction. The TnI115-131 binding site on N-TnC was determined by mapping of chemical shift changes onto the N-TnC NMR structure and was demonstrated to be in the "hydrophobic pocket" (Gagné, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Nat. Struct. Biol. 2, 784-789).

Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study.

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has recently been the target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. We have successfully cloned and bacterially expressed a 15N-labeled PAK pilin peptide spanning residues 128-144 of the intact PAK pilin protein, PAK 128-144(Hs145), and have determined the solution secondary structure of this peptide using heteronuclear multidimensional NMR spectroscopy. The oxidized recombinant peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around the Ile138-Pro139 peptide bond. The pattern of NOEs, temperature coefficients, and coupling constants observed for the trans isomer demonstrate the presence of a type I beta-turn and a type II beta-turn spanning Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively. This is in agreement with the NMR solution structure of the trans isomer of a synthetic PAK 128-144 peptide which showed a type I and a type II beta-turn in these same regions of the sequence [McInnes, C., Sönnichsen, F. D., Kay, C. M., Hodges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hodges, R. S., and Sykes, B. D. (1995) Biochemistry 34, 16255-16268]. The pattern of NOEs, temperature coefficients, and coupling constants observed for the cis isomer also demonstrate a type II beta-turn spanning Pro139-Lys-Gly-Cys142, but suggest a second beta-turn spanning Asp132-Gln-Asp-Glu135. Thus, the cis isomer may also possess a double-turn motif (like the trans isomer), but with different spacing between the turns and a different placement of the first turn in the sequence. The discovery of a double-turn motif in the trans (and cis) recombinant PAK pilin peptide is an extremely important result since the double turn has been implicated as a structural requirement for the recognition of both receptor and antibody. These results pave the way for future isotope-edited NMR studies of the labeled recombinant PAK pilin peptide bound to antibody and receptor, studies integral to the design of an effective synthetic peptide vaccine.

Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction.

To investigate the functional importance of the C-terminal residues 116 to 148 of troponin I (TnI) in the Ca2+-dependent regulation of vertebrate skeletal muscle contraction, we have prepared several synthetic TnI peptide analogs corresponding to various regions within residues 96 to 148 of rabbit skeletal TnI, and analyzed each of these peptides in reconstituted thin filament assays. Our results show that the TnI peptide 96 to 148 (TnI96-148) constitutes the minimal sequence of TnI capable of mediating an inhibitory activity similar to that of intact TnI protein. Truncation of residues 140 to 148 from this region (TnI96-139) or substitution of residues K141, K142 and K144 with alanine (TnI96-148A2) completely abolishes the enhanced inhibitory effect of this region when compared with TnI96-115. A synthetic peptide, residues 128 to 148 of TnI, containing residues 140 to 148, now termed the "second actin-tropomyosin (actin-Tm) binding site" is able to bind specifically to the actin-Tm filament and can induce a weak inhibitory activity on its own. Residues 116 to 131 of TnI do not appear to be important for inhibition, but are critical for interacting with troponin C (TnC). Specific investigations into this region have shown that residues 116 to 126, located directly adjacent to the "inhibitory region" (residues 96 to 115), are critical for allowing TnC to neutralize fully and rapidly the acto-S1-Tm inhibition caused by the various TnI peptides. Furthermore, residues 116 to 131 of TnI, now termed the "second TnC binding site", can significantly enhance the binding affinity of the inhibitory region, residues 96 to 115, for TnC in a Ca2+-dependent manner as determined by affinity chromatography analysis. The implication that TnI residues 116 to 131 bind to the N domain of TnC, and thus the inhibitory region (residues 96 to 115) binds to the C domain of TnC, has made us re-investigate the structural/functional role of the NH2-terminal region of TnI. Studies of competition between the N terminus of TnI (Rp1-40, residues 1 to 40) with the C-terminal peptides TnI96-115, TnI96-131 and TnI96-148 showed that only TnI96-115 could be easily displaced from TnC. These results thus suggest that Ca2+ binding to the regulatory sites of TnC (N domain) alters the binding affinity between the NH2 terminus and the C terminus of TnI for TnC, i.e. a Ca2+-dependent switch between these two sites of TnI for the C domain of TnC. These results have been incorporated into a general model describing the Ca2+-dependent regulation of muscle contraction.

Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament.

The regions of troponin I (TnI) responsible for Ca2+-dependent activation and Ca2+ sensitivity of the actin-myosin subfragment 1-tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATPase assay at pH 7.8 has been applied to reconstituted skeletal muscle thin filaments at actin:S1:TM ratios of 6:1:2. Several TnI fragments (TnI-(104-115), TnI-(1-116), and TnI-(96-148)) and TnI mutants with single amino acid substitutions within the inhibitory region (residues 104-115) were assayed to determine their roles on the regulatory function of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly shown by the reduced potency of TnI mutants with single amino acid substitutions within this region. However, the function of the inhibitory region is modulated by other regions of TnI as observed by the poor inhibitory activity of TnI-(1-116) and the increased potency of the inhibitory region by TnI-(96-148). The regulatory complex composed of TnI-(96-148) plus troponin T-troponin C complex (TnT.C) displays the same Ca2+ sensitivity (pCa50) as intact troponin (Tn) or TnI plus TnT.C while those regulatory complexes composed of TnT.C plus either TnI-(104-115) or TnI-(1-116) had an increase in their pCa50 values. This indicates that the Ca2+ sensitivity or responsiveness of the thin filament is controlled by TnI residues 96-148. The ability of Tn to activate the acto-S1-TM ATPase activity in the presence of calcium to the level of the acto-S1 rate was mimicked by the regulatory complex composed of TnI-(1-116) plus TnT.C and was not seen with complexes composed with either TnI-(104-115) or TnI-(96-148). This indicates that the N terminus of TnI in conjunction with TnT controls the degree of activation of the ATPase activity. Although the TnI inhibitory region (104-115) is the Ca2+-sensitive switch which changes binding sites from actin-TM to TnC in the presence of calcium, its function is modulated by both the C-terminal and N-terminal regions of TnI. Thus, distinct regions of TnI control different aspects of Tn's biological function.

Demonstration of coiled-coil interactions within the kinesin neck region using synthetic peptides. Implications for motor activity.

Kinesin is a dimeric motor protein that can move for several micrometers along a microtubule without dissociating. The two kinesin motor domains are thought to move processively by operating in a hand-over-hand manner, although the mechanism of such cooperativity is unknown. Recently, a approximately 50-amino acid region adjacent to the globular motor domain (termed the neck) has been shown to be sufficient for conferring dimerization and processive movement. Based upon its amino acid sequence, the neck is proposed to dimerize through a coiled-coil interaction. To determine the accuracy of this prediction and to investigate the possible function of the neck region in motor activity, we have prepared a series of synthetic peptides corresponding to different regions of the human kinesin neck (residues 316-383) and analyzed each peptide for its respective secondary structure content and stability. Results of our study show that a peptide containing residues 330-369 displays all of the characteristics of a stable, two-stranded alpha-helical coiled-coil. On the other hand, the NH2-terminal segment of the neck (residues approximately 316-330) has the capacity to adopt a beta-sheet secondary structure. The COOH-terminal residues of the neck region (residues 370-383) are not alpha-helical, nor do they contribute significantly to the overall stability of the coiled-coil, suggesting that these residues mark the beginning of a hinge located between the neck and the extended alpha-helical coiled coil stalk domain. Interestingly, the two central heptads of the coiled-coil segment in the neck contain conserved, "non-ideal" residues located within the hydrophobic core, which we show destabilize the coiled-coil interaction. These residues may enable a portion of the coiled-coil to unwind during the mechanochemical cycle, and we present a model in which such a phenomenon plays an important role in kinesin motility.

Engineering a de novo-designed coiled-coil heterodimerization domain off the rapid detection, purification and characterization of recombinantly expressed peptides and proteins.

Using the techniques of genetic engineering and the principles of protein de novo design, we have developed a unique affinity matrix protein tag system as a rapid, convenient and sensitive method to detect, purify and characterize newly expressed recombinant peptides or proteins from cell extracts. The method utilizes two de novo-designed linear peptide sequences that can selectively dimerize to form the stable protein motif, the two-stranded alpha-helical coiled-coil. In this method, a recombinant bacterial expression vector pRLDE has been engineered so that one of the dimerization strands (E-coil) is expressed as a C-terminal fusion tag on newly expressed peptides or proteins, while the other (K-coil) is either biotin-labeled for detection in a Western blot-type format or immobilized on an insoluble silica support for selective dimerization affinity chromatography. Recombinantly expressed peptides from Escherichia coli containing the dimerization tag have been produced, detected and purified using this method. The recombinant peptides were easily and clearly identified using the biotin-labeled coil, while the single-step affinity purification results indicated the purity of the affinity purified expressed peptides to be > 95%, as assessed by reversed-phase chromatography. The stability of the dimerization domain also allows for the purified peptide to be left attached to the matrix, thus creating a new peptide-bound column that can be used to study peptide-protein or peptide-ligand interactions. Therefore this system offers a new alternative to existing peptide or protein fusion tags and demonstrates the utility of a de novo-designed system.

Immunogenicity of synthetic peptides of Haemophilus influenzae type b outer membrane protein P1.

To identify the B- and T-cell epitopes of P1 of Haemophilus influenzae type b, 13 peptides covering 90% of the protein were chemically synthesized. Mouse, guinea pig, and rabbit antisera raised against purified native P1 were tested for their reactivities against the peptides in peptide-specific enzyme-linked immunosorbent assays (ELISAs). Six immunodominant linear B-cell epitopes were mapped to residues 103 to 137, 189 to 218, 248 to 283, 307 to 331, 384 to 412, and 400 to 437 of the mature P1 protein. When P1 peptides were screened for their reactivities with three human convalescent-phase serum specimens, peptides corresponding to residues 39 to 64, 226 to 253, and 400 to 437 reacted strongly with the antisera. Four regions (residues 39 to 64, 226 to 253, 339 to 370, and 400 to 437) contained murine T-cell epitopes. Rabbit antipeptide antisera were tested for their reactivities with the immunizing peptides and P1 protein by ELISA and immunoblots. All anti-P1 peptide antisera except those raised against peptide HIBP1-8 (residues 279 to 312) or HIBP1-8-keyhole limpet hemocyanin conjugate were shown to be specific for their respective immunizing peptides by ELISA. In addition, rabbit antisera raised against the synthetic peptides corresponding to residues 1 to 29, 39 to 64, 103 to 137, 189 to 218, 226 to 253, 248 to 283, 307 to 331, and 400 to 437 of the mature P1 protein recognized the P1 protein from both typeable and nontypeable isolates. These results suggest that these peptides contain epitopes highly conserved among typeable and nontypeable strains of H. influenzae. However, none of the antipeptide antisera have bactericidal activity, nor were they protective against H. influenzae type b in the infant rat model of bacteremia.