Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence.

The 70-residue carboxyl-terminal domain of the muscle contractile protein troponin-C contains two helix-loop-helix calcium (Ca)-binding sites that are related to each other by approximate twofold rotational symmetry. Hydrophobic residues from the helices and a short three residue beta sheet at the interface of the two sites act to stabilize the protein domain in the presence of Ca. A synthetic 34-residue peptide representing one of these sites (site III) has been synthesized and studied by H-1 nuclear magnetic resonance (NMR) spectroscopy. In solution this peptide undergoes a Ca-induced conformational change to form the helix-loop-helix Ca-binding motif. Two-dimensional nuclear Overhauser effect spectra have provided evidence for the formation of a beta sheet and interactions between several hydrophobic residues from opposing helices as found in troponin-C. It is proposed that a symmetric two-site dimer similar in tertiary structure to the carboxyl-terminal domain of troponin-C forms from the assembly of two site III peptides in the Ca-bound form.

Analysis of the role of antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex virus infection with antibodies to synthetic peptides of glycoprotein D and monoclonal antibodies to glycoprotein B.

The role of antibody in neonatal herpes simplex virus (HSV) infection remains controversial. A battery of well-characterized monoclonal antibodies to HSV glycoprotein B (gB), and polyclonal antibodies against synthetic peptides of predicted epitopes of HSV glycoprotein D (gD) were used to determine in vitro functional activity and association with protection against lethal infection in a murine model of neonatal HSV disease. Antiviral neutralization activity of HSV was not associated with antibody-dependent cellular cytotoxicity (ADCC) activity to HSV-infected cells in vitro. In a model of high dose challenge (10(4) PFU), protection was not afforded by any antibody alone, but was by antibody plus human mononuclear cells, and highly associated with ADCC functional activity (P less than 0.001). In a low dose challenge model, neutralizing activity of antibody alone was associated with protection in vivo (P less than 0.001). Of the nine neutralizing epitopes of gD in vitro, eight were predicted surface regions. Four of the five epitopic sites of gD (2-21, 267-276, 288-297, and 303-312) that were determined to be important targets of ADCC and in vivo protection were also predicted to be surface regions. The only exception was the antiserum to region 52-61 which was predicted to be buried and also showed these activities. ADCC as well as neutralizing antibody activity are important in protection against neonatal HSV infection.

Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria.

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.

Context-dependent effects on the hydrophilicity/hydrophobicity of side-chains during reversed-phase high-performance liquid chromatography: Implications for prediction of peptide retention behaviour.

The present study set out to investigate whether observed relative hydrophilicity/hydrophobicity values of positively charged side-chains (with Lys and Arg as representative side-chains) or hydrophobic side-chains (with Ile as the representative side-chain) were context-dependent, i.e., did such measured values vary depending on characteristics of the peptides within which such side-chains are substituted (overall peptide hydrophobicity, number of positive charges) and/or properties of the mobile phase (anionic counterions of varying hydrophobicity and concentration)? Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to two series of four synthetic peptide analogues (+1, +2, +3 and +4 net charge), the only difference between the two peptide series being the substitution of one hydrophobic Ile residue for a Gly residue, in the presence of anionic ion-pairing reagents of varying hydrophobicity (HCOOH approximately H3PO4 < TFA < PFPA < HFBA) and concentration (2-50 mM). RP-HPLC of these peptide series revealed that the relative hydrophilicity of Lys and Arg side-chains in the peptides increased with peptide hydrophobicity. In addition the relative hydrophobicity of Ile decreased dramatically with an increase in the number of positive charges in the peptide, this hydrophobicity decrease being of greater magnitude as the hydrophobicity of the anionic ion-pairing reagent increased. These results have significant implications in the prediction of peptide retention times for proteomic applications.

Liquid-phase method for peptide synthesis utilizing photolytic cleavage from a new o-nitrobenzoyl polyethylene glycol support.

Photolysis as a method for removal of a tert-butyloxycarbonyl-protected peptide possessing a free C-terminal carboxyl group from a polystyrene support in the solid-phase method of peptide synthesis was introduced by Rich, D. H. and Gurwara, S. K.((1975) J. Am. Chem. Soc. 97, 1575-1578). Using this basic concept we prepared a photosensitive 3-nitro-4-bromomethylbenzoyl polyethylene glycol support for use in the liquid-phase method of peptide synthesis. Photolytic cleavage of a protected tetrapeptide possessing a free C-terminal carboxyl group from the polyethylene glycol support resulted in a 98% yield compared with a 69% yield for the photolytic leavage from the polystyrene support. This application of photolysis as a cleavage method in liquid-phase peptide synthesis avoids the low yields and rather drastic conditons needed to remove a peptide attached directly to the poly-ethylene glycol support in the conventional liquid-phase method.

Interaction of troponin I and troponin C: use of the two-dimensional transferred nuclear Overhauser effect to determine the structure of a Gly-110 inhibitory troponin I peptide analog when bound to cardiac troponin C.

The structure of a peptide analog of the inhibitory region of cardiac troponin-I (N-acetyl-G110-TnI(104-115) amide) when bound to cardiac troponin-C has been determined by 2-dimensional 1H-NMR techniques. The bound structure determined for this peptide is similar to that determined previously for the skeletal peptide (which has a proline at position 110) bound to skeletal troponin-C (Campbell and Sykes (1991) J. Mol. Biol. 222, 405-421). This structure shows a helical like peptide backbone 'bent' around P109-G110 to bring the hydrophobic residues F106, L111 and V114 closer together. The other 'side' of this structure is surrounded by the basic residues extending outwards towards the protein or solution. While the bound structures of the cardiac and skeletal peptides are shown to be quite similar, the cardiac peptide appears more flexible near the central glycine residue.

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes.

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes.

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.

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.

Are trigger sequences essential in the folding of two-stranded alpha-helical coiled-coils?

The amino acid residues comprising the interface between strands of the coiled-coil motif are usually hydrophobic and make a major contribution to coiled-coil folding and stability. However, in some cases the presence of excellent hydrophobic residues at the coiled-coil interface is insufficient for folding. It has been proposed that a "consensus trigger sequence" exists that is necessary within the coiled-coil domains of various proteins to trigger folding. Therefore, in this study we designed a 31-residue hybrid sequence based on sequences from the two-stranded parallel coiled-coil domains of the yeast transcriptional activator GCN4 and the actin-bundling protein Dictyostelium discoideum cortexillin I. The hybrid and its analogs were studied by CD spectroscopy and analytical ultracentrifugation. The hybrid had stable residues in the core "a" and "d" positions in the 3-4 hydrophobic repeat, denoted (abcdefg)n, but did not have a consensus trigger sequence and did not possess appreciable secondary structure as determined by CD spectroscopy. The substitutions in the parent peptide were introduced at positions other than "a" and "d", altering a variety of interactions including alpha-helical propensity, interchain and intrachain electrostatics, and hydrophobicity. Although the substitutions did not bring the overall sequence in closer agreement to the consensus trigger sequence, they increased coiled-coil folding and stability. Therefore, our results suggest that the combination of stabilizing effects along a protein sequence is a more general indicator of protein folding in coiled-coils than the identification of a specific trigger sequence. We propose that surpassing a critical threshold stability value using any type or combination of stabilizing effects will allow coiled-coils to fold, in the absence of a specific trigger sequence per se.

Ion pairs significantly stabilize coiled-coils in the absence of electrolyte.

We have used a synthetic coiled-coil peptide model system to address the long perplexing issue as to why coiled-coils are in general more stable at acidic pH than at neutral pH. Contrary to the above expectation, our results show that at low ionic strength (10 mM) the coiled-coil was much more stable at neutral pH than at acidic pH against both thermal and urea unfolding, indicating that the Lys(+)-Glu- ions pairs present around the coiled-coil interface at neutral pH contribute significantly to the stability of the coiled-coil. However, while the addition of NaCl had no significant effect on the coiled-coil stability at neutral pH, its stability at acidic pH increased dramatically. The cross-over point between the stability at acidic pH and neutral pH occurred at around 100 mM salt, above which the coiled-coil became more stable at acidic pH, in agreement with published results. Therefore, salt effect, rather than intrinsic property, such as carboxyl-carboxyl hydrogen bonding, causes this coiled-coil to become more stable at acidic pH. The preferential stabilizing effect of salt on the coiled-coil at acidic pH can be best explained in terms of the condensation of anions to the positively charged groups on the coiled-coil, the net density of which increases as glutamic acid residues become protonated in acidic pH.

The role of interhelical ionic interactions in controlling protein folding and stability. De novo designed synthetic two-stranded alpha-helical coiled-coils.

The role of interchain ionic interactions in controlling protein folding and stability has been studied by using de novo designed synthetic two-stranded alpha-helical coiled-coils. The model coiled-coil (denoted as EK) consists of two identical 35-residue polypeptide chains with a heptad repeat KgLaGbAcLdEeKf and a Cys residue at position 2 and an Ala residue at position 16 in each chain. The Lys residues at positions "g" in one chain and Glu residues at positions "e" in the other chain are expected to form interchain ion-pairs in the coiled-coil structure. This peptide forms a stable coiled-coil structure in benign medium (50 mM KCl, 25 mM PO4, pH7) with a [urea]1/2 value of 3.5 M. In contrast, two peptide analogs EE (EgLaGbAcLdEeKf) and KK (KgLaGbAcLdKeEf), which differ from EK in that EE contains only negatively charged Glu residues and KK contains only positively charged Lys residues at both positions e and g, each show a random coil structure in benign buffer. However, peptide EE or KK can form a stable coiled-coil structure if the interchain ionic repulsions are effectively suppressed either by changing pH or by using high salt concentrations. An equimolar mixture of these two peptides displays 100% alpha-helical content under the same conditions. These results demonstrate that although the interhelical ionic attractions are not essential for coiled-coil formation, a large number of these weak interactions can play an important role in the assembly of helices. Though interhelical ionic repulsions destabilize the homo-stranded coiled-coil, electrostatic attractions may stabilize the hetero-stranded coiled-coil. In addition, this study also suggests that the folding process for these synthetic model coiled-coils does not involve a single-stranded alpha-helix as a significantly populated folding intermediate.

Salt effects on protein stability: two-stranded alpha-helical coiled-coils containing inter- or intrahelical ion pairs.

An investigation into the role of surface-accessible ion pairs in protein stability was carried out by determining the effects of added salt (KCl, MgCl2 and LaCl3) at neutral and acidic pH on the stability of de novo designed two-stranded alpha-helical coiled-coils. The effects of salt on the stability of coiled-coils containing interhelical i to i' + 5 or intrahelical i to i + 4 and i to i + 3 Lys-Glu ion pairs were compared to the effects on the stability of a control coiled-coil, which contained no intra- or interhelical ion pair. These studies indicate that ionic interactions contribute to coiled-coil stability. The results show that added salt can have complex effects on protein stability, involving stabilizing and destabilizing contributions with the net effect depending on the nature of the charged residues and ionic interactions present in the protein.

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.

Orientation, positional, additivity, and oligomerization-state effects of interhelical ion pairs in alpha-helical coiled-coils.

The role of interhelical g-e' ion pairs in the dimerization specificity and stability of alpha-helical coiled-coils is highly controversial. Synthetic 35-residue coiled-coils based on the heptad repeat QgVaGbAcLdQeK f were used to investigate the effect of orientation of interhelical ion pairs between lysine and glutamic acid residues on coiled-coil stability. Stability was estimated from urea denaturation at 20 degreesC, monitoring unfolding with circular-dichroism spectroscopy. Double mutant cycles were employed to estimate the net interaction energy, Delta DeltaGuint, for the two orientations of the ion pair; Ee-Kg and Ke-Eg. Delta DeltaGuint was found to be about 1.4-fold higher for the Ee-Kg orientation in a coiled-coil containing an N-terminal disulfide bridge. The Delta DeltaGuint value was similar whether obtained from the middle heptad or averaged over all five heptads of the coiled-coil, suggesting that ion pairs contribute additively to coiled-coil stability. The effect of uncompensated charges was also illustrated by single substitutions of Gln with either Lys or Glu, resulting in Lys-Gln or Glu-Gln g-e' pairs. These substitutions were found to be twice as destabilizing at position g as at position e, and Lys was about twice as destabilizing as Glu at both positions e and g. In the absence of an interhelical disulfide bridge, Glu and Lys substitutions in the middle heptad were equally destabilizing at positions e and g (Lys continued to be more destabilizing than Glu) and the Delta DeltaGuint value for Lys-Glu ion pairs was not orientation dependent. These and previous results suggest the non-covalently-linked synthetic coiled-coils behave as molten globules, whereas a disulfide-bridge may "lock in" the structural differences between positions of the heptad repeat. Interhelical Lys-Glu ion pairs in either orientation promoted the formation of trimeric coiled-coils (in the absence of a disulfide bridge) while Gln-Gln g-e' interactions led to dimer formation. The results support a role for g-e' ionic attractions in controlling coiled-coil specificity, stability and oligomerization state, possibly through effects on the side-chain packing at the subunit interface.

Use of a conformationally restricted secondary structural element to display peptide libraries: a two-stranded alpha-helical coiled-coil stabilized by lactam bridges.

A model for an alpha-helical peptide library based on a lactam bridged stabilized two-stranded alpha-helical coiled-coil is described. Sites for library display were incorporated in the middle of the peptide sequence of the most solvent accessible sites of a coiled-coil. A comparison was made between this coiled coil and a native coiled-coil based on the same sequence but lacking the lactam bridges. A lactam bridged peptide where the hydrophobic repeat consisted of all alanine residues, such that the tendency to dimerize would be diminished, was also prepared. This enabled us to determine the role tertiary interactions play in maintaining library positions in a helical conformation. The consensus sequence derived from a Zn finger library screened against an IgA reactive against the lipopolysaccharide of Shigella flexneri was transplanted into the peptides. CD spectroscopy revealed that although both coiled-coils are highly helical at 100 microM, the lactam bridge enhanced dimerization and allow the peptide to maintain its coiled-coil conformation at lower peptide concentrations. The helical content of the alanine based peptide was 69% and was independent of concentration over a range of 1.4 to 1410 microM. Urea denaturation studies indicated that the coiled-coils were considerably more stable than the alanine based peptide and that the lactam bridge coiled-coil was more stable than the native coiled coil by 1.6 kcal/mol. The lactam bridged coiled-coil was found to inhibit binding of the Zn finger peptide to the IgA in a concentration dependent manner with an IC50 of 5.0 microM whereas the peptide lacking the lactam bridges was much less effective in inhibiting binding. The alanine peptide was less active than the lactam bridged coiled-coil but more effective than the native coiled-coil with an IC50 of 16 microM. The versatility of the lactam stabilized coiled-coil template was demonstrated by incorporating five Gly residues into the library display sites. While the native coiled-coil adopted a random conformation the lactam bridged coiled-coil was 59% helical. Incorporation of a Cys-Gly-Gly linker to the N terminus and formation of a disulfide bond stabilized the peptide to the extent that it adopted a highly helical coiled-coil (94%) with a [urea]1/2 value of 5.9 M. Since the five glycine residues represent one of the most destabilizing combinations of amino acids that would be encountered at the five library display sites (with the exception of Pro), this stabilized coiled-coil should maintain its folded conformation regardless of the amino acids occupying the library positions.

The entericidin locus of Escherichia coli and its implications for programmed bacterial cell death.

Antidote/toxin gene pairs known as "addiction modules" can maintain plasmids in bacterial populations by means of post-segregational killing. However, several chromosome-encoded addiction modules may provide an entirely distinct function in the programmed cell death of moribund subpopulations under starvation conditions. We now report a novel chromosomal bacteriolytic module of Escherichia coli called the entericidin locus, which is activated in stationary phase under high osmolarity conditions by sigmaS and simultaneously repressed by the osmoregulatory EnvZ/OmpR signal transduction pathway. The entericidin locus encodes tandem paralogous genes (ecnAB) and directs the synthesis of two small cell-envelope lipoproteins. An attenuator precedes ecnA and an ompR-sensitive sigmaS promoter governs expression of ecnB. The entericidin A lipoprotein is an antidote to the bacteriolytic lipoprotein entericidin B. The entericidins are predicted to adopt amphipathic alpha-helical structures and to reciprocally modulate membrane stability. The entericidin locus is not present on any known plasmids, but is conserved in the homologous region of the Citrobacter freundii chromosome. Although the cloned C. freundii entericidin locus is expressed in E. coli independently of ompR, it carries an additional ompR-like gene called ecnR. The organization of the entericidin locus as a chromosomal antidote/toxin gene pair, which is regulated by both positive and negative osmotic signals during starvation, is consistent with an emerging paradigm of programmed bacterial cell death.

Insights into the mechanism of heterodimerization from the 1H-NMR solution structure of the c-Myc-Max heterodimeric leucine zipper.

The oncoprotein c-Myc (a member of the helix-loop-helix-leucine zipper (b-HLH-LZ) family of transcription factors) must heterodimerize with the b-HLH-LZ Max protein to bind DNA and activate transcription. It has been shown that the LZ domains of the c-Myc and Max proteins specifically form a heterodimeric LZ at 20 degreesC and neutral pH. This suggests that the LZ domains of the c-Myc and Max proteins are playing an important role in the heterodimerization of the corresponding gene products in vivo. Initially, to gain an insight into the energetics of heterodimerization, we studied the stability of N-terminal disulfide-linked versions of the c-Myc and Max homodimeric LZs and c-Myc-Max heterodimeric LZ by fitting the temperature-induced denaturation curves monitored by circular dichroism spectroscopy. The c-Myc LZ does not homodimerize (as previously reported) and the c-Myc-Max heterodimeric LZ is more stable than the Max homodimeric LZ at 20 degreesC and pH 7.0. In order to determine the critical interhelical interactions responsible for the molecular recognition between the c-Myc and Max LZs, the solution structure of the disulfide-linked c-Myc-Max heterodimeric LZ was solved by two-dimensional 1H-NMR techniques at 25 degreesC and pH 4.7. Both LZs are alpha-helical and the tertiary structure depicts the typical left-handed super-helical twist of a two-stranded parallel alpha-helical coiled-coil. A buried salt bridge involving a histidine on the Max LZ and two glutamate residues on the c-Myc LZ is observed at the interface of the heterodimeric LZ. A buried H-bond between an asparagine side-chain and a backbone carbonyl is also observed. Moreover, evidence for e-g interhelical salt bridges is reported. These specific interactions give insights into the preferential heterodimerization process of the two LZs. The low stabilities of the Max homodimeric LZ and the c-Myc-Max heterodimeric LZ as well as the specific interactions observed are discussed with regard to regulation of transcription in this family of transcription factors.

Structural predictions of AgfA, the insoluble fimbrial subunit of Salmonella thin aggregative fimbriae.

The unusually stable and multifunctional, thin aggregative fimbriae common to all Salmonella spp. are principally polymers of the fimbrin subunit, AgfA. AgfA of Salmonella enteritidis consists of two domains: a protease-sensitive, 22 amino acid residue N-terminal region and a protease-resistant, 109 residue C-terminal core. The unusual amino acid sequence of the AgfA core region comprises two-, five- and tenfold internal sequence homology patterns reflected in five conserved, 18-residue tandem repeats. These repeats have the consensus sequence, Sx5QxGx2NxAx3Q and are linked together by four or five residues, (x)xAx2. The predicted secondary structure for this unusual arrangement of tandem repeats in AgfA indicates mainly extended conformation with the beta strands linked by four to six residues. Candidate proteins of known structure with motifs of alternating beta strands and short loops were selected from folds described in SCOP as a source of coordinates for AgfA model construction. Three all-beta class motifs selected from the Serratia marcescens metalloprotease, myelin P2 protein or vitelline membrane outer protein I were used for initial AgfA homology build-up procedures ultimately resulting in three structural models; beta barrel, beta prism and parallel beta helix. The beta barrel model is a compact, albeit irregular structure, with the beta strands arranged in two antiparallel beta sheet faces. The beta prism model does not reflect the 5 or 10-fold symmetry of the AgfA primary sequence. However, the favored, parallel beta helix model is a compact coil of ten helically arranged beta strands forming two parallel beta sheet faces. This arrangement predicts a regular, potentially stable, C-terminal core region consistent with the observed tandem repeat sequences, protease-resistance and strong tendency of this fimbrin to oligomerize and aggregate. Positional conservation of amino acid residues in AgfA and the Escherichia coli AgfA homologue, CsgA, provides strong support for this model. The parallel beta helix model of AgfA offers an interesting solution to a multifunctional fimbrin molecular surface having solvent exposed areas, regions for major and minor subunit interactions as well as fiber-fiber interactions common to many bacterial fimbriae.

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.