Coiled-coil Structural Motif Research Articles

Design of Broadly Cross-Reactive M Protein-Based Group A Streptococcal Vaccines.

Group A streptococcal infections are a significant cause of global morbidity and mortality. A leading vaccine candidate is the surface M protein, a major virulence determinant and protective Ag. One obstacle to the development of M protein-based vaccines is the >200 different M types defined by the N-terminal sequences that contain protective epitopes. Despite sequence variability, M proteins share coiled-coil structural motifs that bind host proteins required for virulence. In this study, we exploit this potential Achilles heel of conserved structure to predict cross-reactive M peptides that could serve as broadly protective vaccine Ags. Combining sequences with structural predictions, six heterologous M peptides in a sequence-related cluster were predicted to elicit cross-reactive Abs with the remaining five nonvaccine M types in the cluster. The six-valent vaccine elicited Abs in rabbits that reacted with all 11 M peptides in the cluster and functional opsonic Abs against vaccine and nonvaccine M types in the cluster. We next immunized mice with four sequence-unrelated M peptides predicted to contain different coiled-coil propensities and tested the antisera for cross-reactivity against 41 heterologous M peptides. Based on these results, we developed an improved algorithm to select cross-reactive peptide pairs using additional parameters of coiled-coil length and propensity. The revised algorithm accurately predicted cross-reactive Ab binding, improving the Matthews correlation coefficient from 0.42 to 0.74. These results form the basis for selecting the minimum number of N-terminal M peptides to include in potentially broadly efficacious multivalent vaccines that could impact the overall global burden of group A streptococcal diseases.

Transmembrane Prolines Mediate Signal Sensing and Decoding in Bacillus subtilis DesK Histidine Kinase.

Environmental awareness is an essential attribute of all organisms. The homeoviscous adaptation system of Bacillus subtilis provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense the order of membrane lipids with the transmembrane (TM) protein DesK, which has an N-terminal sensor domain and an intracellular catalytic effector domain. DesK exhibits autokinase activity as well as phosphotransferase and phosphatase activities toward a cognate response regulator, DesR, that controls the expression of an enzyme that remodels membrane fluidity when the temperature drops below ∼30°C. Membrane fluidity signals are transmitted from the DesK sensor domain to the effector domain via rotational movements of a connecting 2-helix coiled coil (2-HCC). Previous molecular dynamic simulations suggested important roles for TM prolines in transducing the initial signals of membrane fluidity status to the 2-HCC. Here, we report that individual replacement of prolines in DesKs TM1 and TM5 helices by alanine (DesKPA) locked DesK in a phosphatase-ON state, abrogating membrane fluidity responses. An unbiased mutagenic screen identified the L174P replacement in the internal side of the repeated heptad of the 2-HCC structure that alleviated the signaling defects of every transmembrane DesKPA substitution. Moreover, substitutions by proline in other internal positions of the 2-HCC reestablished the kinase-ON state of the DesKPA mutants. These results imply that TM prolines are essential for finely tuned signal generation by the N-terminal sensor helices, facilitating a conformational control by the metastable 2-HCC domain of the DesK signaling state.IMPORTANCE Signal sensing and transduction is an essential biological process for cell adaptation and survival. Histidine kinases (HK) are the sensory proteins of two-component systems that control many bacterial responses to different stimuli, like environmental changes. Here, we focused on the HK DesK from Bacillus subtilis, a paradigmatic example of a transmembrane thermosensor suited to remodel membrane fluidity when the temperature drops below 30°C. DesK provides a tractable system for investigating the mechanism of transmembrane signaling, one of the majors interrogates in biology to date. Our studies demonstrate that transmembrane proline residues modulate the conformational switch of a 2-helix coiled-coil (2-HCC) structural motif that controls input-output in a variety of HK. Our results highlight the relevance of proline residues within sensor domains and could inspire investigations of their role in different signaling proteins.

Stabilizing Protein Motifs with a Genetically Encoded Metal-Ion Chelator

The N-peptide of HIV gp41 forms a trimeric coiled-coil intermediate during host cell-viral fusion. Stable mimics of this coiled coil could potentially serve as HIV vaccine candidates or inhibitors of viral entry. Therefore, a variety of approaches have been investigated to maintain the N-peptide in its trimeric helical conformation. Here, we utilize a genetic method to incorporate the metal chelating noncanonical amino acid (2,2'-bipyridin-5-yl)alanine (BpyAla) into IZN17, an established trimeric coiled-coil gp41 model. Wedemonstrate that BpyAla-IZN17 acquires Fe(II) during expression in Escherichia coli, resulting in the formation of a highly stable IZN17 helical trimer with a Tm > 95°C. Removal of Fe(II) results in a 27°C decrease in thermal stability. Replacement of Fe(II) with Zn(II) also affords a Tm > 90°C. Thus, the genetic introduction of BpyAla into polypeptide provides astraightforward method for stabilizing coiled-coilstructural motifs in recombinantly engineered proteins.

Tensile Mechanics of Coiled Coil Protein Structures

A coiled-coil protein structure consists of two or more interacting α-helical strands that together form a supercoil structure. Coiled-coil structures entail unique mechanical properties that are critical to the function and integrity of various motor proteins, cytoskeletal filaments and extra-cellular matrix proteins. Here we present a thermodynamic model to predict the mechanical properties of a given coiled-coil structural motif. Within the proposed model we identify and quantify various energetic and entropic effects, responsible for dimerization of two helical polypeptides into a coiled coil structure. We determine our model parameters by examining a large body of solved protein structures that contain coiled coil motifs. This would allow us to develop a thermodynamic model for predicting the propensity of given amino acid sequence to form a coiled coil structure. Further incorporation of the above model into our previously developed α-helix tensile mechanics model would enable us to predict the structural response of a given coiled coil motif to a tensile stress.

Growth Factor Tethering to Protein Nanoparticles via Coiled-Coil Formation for Targeted Drug Delivery.

Protein-based nanoparticles are attractive carriers for drug delivery because they are biodegradable and can be genetically designed. Moreover, modification of protein-based nanoparticles with cell-specific ligands allows for active targeting abilities. Previously, we developed protein nanoparticles comprising genetically engineered elastin-like polypeptides (ELPs) with fused polyaspartic acid tails (ELP-D). Epidermal growth factor (EGF) was displayed on the surface of the ELP-D nanoparticles via genetic design to allow for active cell-targeting abilities. Herein, we focused on the coiled-coil structural motif as a means for noncovalent tethering of growth factor to ELP-D. Specifically, two peptides known to form a heterodimer via a coiled-coil structural motif were fused to ELP-D and single-chain vascular endothelial growth factor (scVEGF121), to facilitate noncovalent tethering upon formation of the heterodimer coiled-coil structure. Drug-loaded growth factor-tethered ELP-Ds were found to be effective against cancer cells by provoking cell apoptosis. These results demonstrate that tethering growth factor to protein nanoparticles through coiled-coil formation yields a promising biomaterial candidate for targeted drug delivery.

Structural Plasticity of Helical Nanotubes Based on Coiled-Coil Assemblies

Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies.

Role of the coiled-coil structural motif in polyglutamine aggregation.

Polyglutamine repeat motifs are known to induce protein aggregation in various neurodegenerative diseases, and flanking sequences can modulate this behavior. It has been proposed that the 17 N-terminal residues (Htt(NT)) of the polyglutamine-containing huntingtin protein accelerate the kinetics of aggregation due to the formation of helix-rich oligomers through helix-pairing interactions. Several hypotheses that explain the role of helical interactions in modulating aggregation have been proposed. These include (1) an increase in the effective concentration of polyglutamine chains (proximity model), (2) the induction of helical structure within the polyglutamine domain itself (transformation model), and/or (3) interdomain interactions between the flanking sequence and the polyglutamine domain (domain cross-talk model). These hypotheses are tested by studying the kinetics of polyglutamine aggregation using a Q25 sequence fused to a well-defined heterotetrameric coiled-coil model system. Using a combined spectroscopic and dye binding approach, it is shown that stable coiled-coil formation strongly inhibits polyglutamine aggregation, suggesting that the proximity and transformation models are insufficient to explain the enhanced aggregation seen in Htt(NT)-polyglutamine constructs. Consistent with other published work, our data support a model in which domain cross-talk prevents formation of a nonspecific aggregated collapsed polyglutamine state, which can act to inhibit conversion to a fibrillar state. Because our model system has a charged to nonpolar residue ratio much higher than that of the Htt(NT) sequence, domain cross-talk is severely weakened, thus favoring the nonspecific aggregation pathway and significantly inhibiting aggregation through a fibrillar pathway.

Thermodynamic Analysis of Self-Assembly in Coiled-Coil Biomaterials

Coiled-coil protein structural motifs have proven amenable to the design of structurally well-defined biomaterials. Mesoscale structural properties can be fairly well predicted based on rules governing the chemical interactions between the helices that define this structural motif. We explore the role of the hydrophobic core residues on the self-assembly of a coiled-coil polymer through a mutational analysis coupled with a salting-out procedure. Because the resultant polymers remain in solution, a thermodynamic approach is applied to characterize the polymer assembly using conventional equations from polymer theory to extract nucleation and elongation parameters. The stabilities and lengths of the polymers are measured using circular dichroism spectropolarimetry, sizing methods including dynamic light scattering and analytical ultracentrifugation, and atomic force microscopy to assess mesoscale morphology. Upon mutating isoleucines at two core positions to serines, we find that polymer stability is decreased while the degree of polymerization is about the same. Differences in results from circular dichroism and dynamic light scattering experiments suggest the presence of a stable intermediate state, and a scheme is proposed for how this intermediate might relate to the monomer and polymer states.

Characterisation of hookworm heat shock factor binding protein (HSB-1) during heat shock and larval activation

When hookworm infective L3s infect their mammalian host, they undergo a temperature shift from that of the ambient environment to that of their endothermic host. Additionally, L3s living in the environment can be exposed to temperature extremes associated with weather fluctuations. The heat shock response (HSR) is a conserved response to heat shock and other stress that involves the expression of protective heat shock proteins (HSPs). The HSR is controlled by heat shock factor-1 (HSF-1), a conserved transcription factor that binds to a heat shock element in the promoter of HSPs, causing their expression. HSF-1 is negatively regulated in part by a HSF binding protein (HSB-1) that binds to and removes HSF-1 trimers bound to HSP gene promoters, resulting in attenuation of the HSR. Herein we describe an HSB-1 orthologue, Ac-HSB-1, from the hookworm Ancylostoma caninum. The Ac-hsb-1 cDNA encodes a 79 amino acid protein that is 71% identical to the Caenorhabditis elegans HSB-1, and is predicted to share the characteristic coiled-coil structural motif comprised of two interacting alpha helices. Recombinant Ac-HSB-1 immunoprecipitated Ce-HSF-1 expressed in mammalian cells that had been heat shocked for 1h at 42°C, but not from cells incubated at 37°C, indicating that HSB-1 only bound to the active DNA binding form of HSF-1. Expression of Ac-hsb-1 transcripts decreased following 1h of heat shock, but increased when L3s were incubated at 37°C for 1h. Activation of hookworm L3s induces a five–sixfold increase in Ac-hsb-1 expression that peaks at 12h, coincident with L3 feeding, but that subsequently decreases to two–threefold above control at 24h. Recombinant Ac-HSB-1 immunoprecipitates greater amounts of 70 and 40kDa proteins from extracts of activated L3s than from non-activated L3s. We propose that an increase in Ac-hsb-1 levels early in activation allows feeding to resume, but that a subsequent decrease in expression permits a HSR that protects non-developing L3s at host-like temperatures. Further investigations of the HSR will clarify the role of HSB-1 and HSF-1 in hookworm infection.

CCHMM_PROF: a HMM-based coiled-coil predictor with evolutionary information

The widespread coiled-coil structural motif in proteins is known to mediate a variety of biological interactions. Recognizing a coiled-coil containing sequence and locating its coiled-coil domains are key steps towards the determination of the protein structure and function. Different tools are available for predicting coiled-coil domains in protein sequences, including those based on position-specific score matrices and machine learning methods. In this article, we introduce a hidden Markov model (CCHMM_PROF) that exploits the information contained in multiple sequence alignments (profiles) to predict coiled-coil regions. The new method discriminates coiled-coil sequences with an accuracy of 97% and achieves a true positive rate of 79% with only 1% of false positives. Furthermore, when predicting the location of coiled-coil segments in protein sequences, the method reaches an accuracy of 80% at the residue level and a best per-segment and per-protein efficiency of 81% and 80%, respectively. The results indicate that CCHMM_PROF outperforms all the existing tools and can be adopted for large-scale genome annotation. The dataset is available at http://www.biocomp.unibo.it/ approximately lisa/coiled-coils. The predictor is freely available at http://gpcr.biocomp.unibo.it/cgi/predictors/cchmmprof/pred_cchmmprof.cgi. piero@biocomp.unibo.it.

Engineering responsive mechanisms to control the assembly of peptide-based nanostructures

Complex biological machines arise from self-assembly on the basis of structural features programmed into sequence-specific macromolecules (i.e. polypeptides and polynucleotides) at the molecular level. As a consequence of the near-absolute control of macromolecular architecture that results from such sequence specificity, biological structural platforms may have advantages for the creation of functional supramolecular assemblies in comparison with synthetic polymers. Thus biological structural motifs present an attractive target for the synthesis of artificial nanoscale systems on the basis of relationships between sequence and supramolecular structure that have been established for native biological assemblies. In the present review, we describe an approach to the creation of structurally defined supramolecular assemblies derived from synthetic alpha-helical coiled-coil structural motifs. Two distinct challenges are encountered in this approach to materials design: the ability to recode the canonical sequences of native coiled-coil structural motifs to accommodate the formation of structurally defined supramolecular assemblies (e.g. synthetic helical fibrils) and the development of methods to control supramolecular self-assembly of these peptide-based materials under defined conditions that would be amenable to conventional processing methods. In the present review, we focus on the development of mechanisms based on guest-host recognition to control fibril assembly/disassembly. This strategy utilizes the latent structural specificity encoded within sequence-defined peptides to couple a conformational transition within the coiled-coil motifs to incremental changes in environmental conditions. The example of a selective metal-ion-induced conformational switch will be employed to validate the design principles.

Intramolecular Charge Interactions as a Tool to Control the Coiled‐Coil‐to‐Amyloid Transformation

Under the influence of a changed environment, amyloid-forming proteins partially unfold and assemble into insoluble beta-sheet rich fibrils. Molecular-level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the alpha-helical coiled-coil structural motif. In all peptides, three valine residues, known to prefer the beta-sheet conformation, have been incorporated at the solvent-exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH-controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted alpha-helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid-like fibrils. Peptide B features a glutamate-based switch functionality and exhibits opposite pH-dependent folding behavior. In this case, alpha-helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.

Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth

The inositol 1,3,4,5,6-pentakisphosphate (IP(5)) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP(6)) in eukaryotic cells. Previous studies have shown that IP(6) is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1(+) in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1(+) deleted (ipk1Delta) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP(6) production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1(+) function, encoding an IP(6) kinase downstream of Ipk1, had an effect on ipk1Delta cells. The asp1Delta mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Delta cells. Moreover, ipk1Delta asp1Delta double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Delta mutant. This suggested novel roles for asp1(+) upstream of ipk1(+). We propose that IP(6) production is a key signaling linchpin for regulating multiple essential cellular processes.

How Metal Ions Affect Amyloid Formation: Cu2+‐ and Zn2+‐Sensitive Peptides

The common feature of proteins involved in many neurodegenerative diseases is their ability to adopt at least two different stable conformations. The conformational transition that shifts the equilibrium from the functional, mostly partially alpha-helical structure, to the beta-sheet rich amyloid can be triggered by numerous factors, such as mutations in the primary structure or changes in the environment. We present a set of model peptides that, without changes in their primary structure, react in a predictable fashion in the presence of transition metal ions by adopting different conformations and aggregate morphologies. These de novo designed peptides strictly follow the characteristic heptad repeat of the alpha-helical coiled-coil structural motif. Furthermore, domains that favor beta-sheet formation have been incorporated to make the system prone to amyloid formation. As a third feature, histidine residues create sensitivity towards the presence of transition metal ions. CD spectroscopy, ThT fluorescence experiments, and transmission electron microscopy were used to characterize peptide conformation and aggregate morphology in the presence of Cu2+ and Zn2+. Furthermore, the binding geometry within peptide-Cu2+ complexes was characterized by electron paramagnetic resonance spectroscopy.

Structure and functional characterization of the periplasmic N-terminal polypeptide domain of the sugar-specific ion channel protein (ScrY porin).

The structure of the sucrose-specific porin (ScrY) from Salmonella typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel beta-strands, associated as a trimer complex similar to ion-transport channels. However, the 71-amino-acid-residue N-terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density. The N-terminal polypeptide contains a coiled-coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin. In this study the proteolytic stability and a specific proteolytic truncation site at the N-terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post-source-decay fragmentation. The secondary structure and supramolecular association of the coiled-coil N-terminal domain were determined by chemical synthesis of the complete N-terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization. Circular dichroism spectra revealed predominant alpha-helical conformation for the putative coiled-coil domain comprising residues 4-46. Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data. The role of the N-terminal domain in sugar binding was examined by comparative TR-NOE-NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(delta1-62), lacking the N-terminal polypeptide. The TR-NOE-NMR data showed a strong influence of ScrY porin on the sugar-binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low-affinity sugar binding.

An engineered leucine zipper a position mutant with an unusual three-state unfolding pathway.

The leucine zipper is a dimeric coiled-coil structural motif consisting of four to six heptad repeats, designated (abcdefg)(n). In the GCN4 leucine zipper, a position 16 in the third heptad is occupied by an Asn residue whereas the other a positions are Val residues. Recently, we have constructed variants of the GCN4 leucine zipper in which the a position Val residues were replaced by Ile. The folding and unfolding of the wild-type GCN4 leucine zipper and the Val to Ile variant both adhere to a simple two-state mechanism. In this study, another variant of the GCN4 leucine zipper was constructed by moving the single Asn residue from a position 16 to a position 9. This switch causes the thermal unfolding of the GCN4 leucine zipper to become three state. The unfolding pathway of this variant was determined by thermal denaturation, limited proteinase K digestion, and sedimentation equilibrium analysis. Our data are consistent with a model in which the variant first unfolds from its N terminus and changes the oligomerization specificity from a native dimer to a partially unfolded intermediate containing a mixture of dimers and trimers and then completely unfolds to unstructured monomers.

New gene family defined by MORC, a nuclear protein required for mouse spermatogenesis.

Mammalian spermatogenesis is a complex developmental process. The analysis of mouse mutations has provided insight into biochemical pathways required for completion of this process. We previously described the autosomal recessive mouse morc TgN(Tyr)1Az(microrchidia) mutation, a serendipitous transgenic insertional mutation which causes arrest of spermatogenesis prior to the pachytene stage of meiosis prophase I. We now report the molecular characterization of the morc locus and positional cloning of a gene disrupted by the morc TgN(Tyr)1Az mutation. This gene, which we term Morc, encodes a 108 kDa protein expressed specifically in male germ cells. The transgene integrated within the first intron of Morc and was accompanied by an intragenic deletion of approximately 13 kb of genomic sequences, removing exons 2-4 and abrogating expression of the wild-type transcript. Analysis of the MORC protein sequence revealed putative nuclear localization signals, two predicted coiled-coil structural motifs and limited homology to GHL (GyraseB, Hsp90, MutL) ATPase. Epitope-tagged MORC protein expressed in COS7 cells localized to the nucleus. We also cloned the human MORC homolog and show that it too is testis-specific, but closely related human genes are transcribed in multiple somatic tissues. Homologous proteins are also present in zebrafish, nematodes, slime mold and plants. Thus, cloning of Morc defines a novel gene family whose members are likely to serve important biological functions in both meiotic and mitotic cells of multicellular organisms.

A distinct 14 residue site triggers coiled-coil formation in cortexillin I.

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.

A synthetic peptide ligase.

The preparation of synthetic molecules showing the remarkable efficiencies characteristic of natural biopolymer catalysts remains a formidable challenge for chemical biology. Although significant advances have been made in the understanding of protein structure and function, the de novo construction of such systems remains elusive. Re-engineered natural enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups, have been successful in catalysing a number of chemical transformations, sometimes with impressive efficiencies. But efforts to produce wholly synthetic catalytic peptides have typically resulted in compounds with questionable structural stability, let alone reactivity. Here we describe a 33-residue synthetic peptide, based on the coiled-coil structural motif, which efficiently catalyses the condensation of two shorter peptide fragments with high sequence- and diastereoselectivity. Depending on the substrates used, we observe rate enhancements of tenfold to 4,100-fold over the background, with catalytic efficiencies in excess of 10(4). These results augur well for the rational design of functional peptides.

Thermodynamic analysis of a designed three-stranded coiled coil.

The study and successful design of coiled-coil protein structural motifs have provided much insight into the rules governing protein folding and stability. In this work we use a thermodynamic approach to quantitate the rules that govern the specific oligomerization of coiled coils. We have designed a highly stable trimeric coiled coil by placing valine residues at each a position and leucine residues at each d position of the heptad repeating unit. The peptide forms a very stable trimer as determined by sedimentation equilibrium, and the concentration dependence of its circular dichroism spectrum follows a cooperative monomer/dimer/trimer equilibrium with the dimer state as a highly unstable intermediate. Its guanidinium chloride denaturation curve was collected at several peptide concentrations, and analysis of the data confirms the cooperativity of the trimerization process and provides a free energy of stabilization of - 18.4 kcal mol-1 for the trimer. The heat capacity, delta Cp, was measured by global analysis of thermal unfolding data collected at a number of guanidinium chloride concentrations. Guanidinium chloride induces cold denaturation in the thermal unfolding curves, providing a reasonably well-determined value for delta Cp of 750 cal deg-1 mol-1. This translates to a delta Cp of 8.6 cal deg-1 mol-1 per residue and corresponds well to that expected of a coiled coil with a well-defined tertiary structure.