Biomedical subjects
Publications and source records attributed to R S Hodges.
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Monoclonal antibodies (MAbs) to a Burkholderia (Pseudomonas) cepacia 36-kDa protease (PSCP) which neutralize PSCP and Pseudomonas aeruginosa elastase but not P. aeruginosa alkaline protease have been isolated (C. Kooi et al., Infect. Immun. 62:2811-2817, 1994). These MAbs, designated 36-6-6 and 36-6-8, react with N-chlorosuccinimide cleavage products of P. aeruginosa elastase, consistent with the recognition of a 13.9-kDa fragment which contains the active site. Overlapping 9-mer peptides that span this region were synthesized. Neutralizing MAbs to PSCP reacted strongly with two peptides (341HGFTEQNSG349 and 395RYM DQPSRD403). Peptide 341HGFTEQNSG349 overlaps the motif 337HEXXH341, which has been found in many zinc-dependent endopeptidases. Peptide 395RYMDQPSRD403 lies between E361, which binds a zinc atom, and H420, which acts as a proton donor at the active site. Polyclonal rabbit sera raised against these peptides reacted with elastase on Western immunoblots and by enzyme-linked immunosorbent assay. With hide powder azure as the substrate, antisera to either HGFTEQNG and RYMDQPSRD completely neutralized the activities of elastase, thermolysin, Vibrio cholerae hemagglutinin/protease, and PSCP but had no effect on P. aeruginosa alkaline protease or the Serratia marcescens major protease. These results suggest that the MAbs recognize two different epitopes on P. aeruginosa elastase and that antibodies raised against synthetic peptides corresponding to either of these epitopes neutralize proteolytic activity.
We have evaluated the effect of ring size of gramicidin S analogs on secondary structure, lipid binding, lipid disruption, antibacterial and hemolytic activity. Cyclic analogs with ring sizes ranging from 4 to 14 residues were designed to maintain the amphipathic character as found in gramicidin S and synthesized by solid phase peptide synthesis. The secondary structure of these peptides showed a definite periodicity in beta-sheet content, with rings containing 6, 10, and 14 residues exhibiting beta-sheet structure, and rings containing 8 or 12 residues being largely disordered. Peptides containing 4 or 6 residues did not bind lipopolysaccharide, whereas longer peptides showed a trend of increasing binding affinity for lipopolysaccharide with increasing length. Destabilization of Escherichia coli outer membranes was only observed in peptides containing 10 or more residues. Peptides containing fewer than 10 residues were completely inactive and exhibited no hemolytic activity. The 10-residue peptide showed an activity profile similar to that of gramicidin S itself, with activity against Gram-positive and Gram-negative microorganisms as well as yeast, but also showed high hemolytic activity. Differential activities were obtained by increasing the size of the ring to either 12 or 14 residues. The 14-residue peptide showed no antibiotic activity but exhibited increased hemolytic activity. The 12-residue peptide lost activity against Gram-positive bacteria, retained activity against Gram-negative microorganisms and yeast, but displayed decreased hemolytic activity. Biological activities in the 12-residue peptide were optimized by a series of substitutions in residues comprising both hydrophobic and basic sites resulting in a peptide that exhibited activities comparable with gramicidin S against Gram-negative microorganisms and yeast but with substantially lower hemolytic activity. Compared with gramicidin S, the best analog showed a 10-fold improvement in antibiotic specificity for Gram-negative microorganisms and a 7-fold improvement in specificity for yeast over human erythrocytes as determined by a therapeutic index. These results indicate that it is possible to modulate structure and activities of cyclic gramicidin S analogs by varying ring sizes and further show the potential for developing clinically useful antibiotics based on gramicidin S.
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 surface plasmon resonance (SPR) technique was used to study the formation kinetics of a de novo designed coiled-coil (E/K coil). The E/K coil is made up of two distinct peptides (E and K) each with five heptad (g-a-b-c-d-e-f) repeats. The E peptide's heptad sequence is E-V-S-A-L-E-K, and the K peptide's heptad sequence is K-V-S-A-L-K-E. A linker C-nL-G-G-G (nL = norleucine) is present at the C-terminus of the E peptide and at the N-terminus of the K peptide for the SPR studies. Heterodimer formation involves both electrostatic and hydrophobic interactions at the dimer interface. Under conditions that favor the heterodimer formation, the CD signal ([theta]222) varied as a function of peptide concentration. The estimated dissociation constant (Kd) was 2.45 +/- 0.71 nM. Denaturation studies with guanidine-HCI (GdnHC11/2 = 3.9 M) suggested a value of 3.53 +/- 0.48 nM. For the SPR investigation, the peptides were biotinylated and linked to streptavidin in order to increase their effective molecular weight and consequently enhance the signal intensity. Biotinylation in itself did not impede coiled-coil formation based on CD measurements. The biosensor study revealed a slow dissociation rate constant for the heterodimer (kd approximately 2 x 10(-4) s-1) and a moderately fast association rate constant [ka approximately (4.27-4.53) x 10(5) M-1 s-1). This gives a calculated Kd of 0.47-0.50 nM, which agrees reasonably well with the equilibrium CD studies. Therefore, based on the SPR data, the preference for heterodimer formation is due to a combination of moderately fast association and slow dissociation rates.
A series of lactam-bridged and linear 14 residue amphipathic alpha-helical peptides based on the sequence Ac-EXEALKKEXEALKK-amide were prepared in order to determine the effect of decreasing the hydrophobicity of the nonpolar face to helical content and stability. This was done by substituting position X by Ile, Val, and Ala. Lactam bridges spaced i to i + 4 were formed between the side chains of Glu3 and Lys7 and Glu10 and Lys14 while the linear noncyclized peptides could potentially form i to i + 4 salt bridges with the same residues. It was found that in all cases the lactam-bridged peptides were substantially more helical than the corresponding linear peptides as determined by CD spectroscopy. Moreover, the helical content approached 100% for the lactam-bridged peptides X = Ile and Ala and was greater than 80% for X = Val. For X = Ile and Val, this was partly due to the ability of the lactam bridges to enhance interchain interactions relative to the linear versions of the same sequence. Size-exclusion chromatography demonstrated that the Ile-based peptide associates as a dimer. The alanine-based lactam-bridged peptide was found to be monomeric as determined by concentration dependency studies and size-exclusion chromatography. Thermal denaturation studies in benign media indicated that the lactam-based peptides were very stable. The conformation of the Ala-based lactam peptide was further characterized by two-dimensional NMR spectroscopy and was found to be highly helical. The results demonstrate the ability of lactam bridges to stabilize the helical conformation and enhance dimerization of peptides based on a 3,4 hydrophobic heptad repeat. The substitution of Ala residues in the hydrophobic face of the alpha-helix can prevent dimerization and specify monomeric helical structure.
The effects of electrostatic interactions on the stability of coiled-coils were investigated using the strategy of shuffling the sequence without changing the overall content of amino acid residues in the peptides. Shuffling the sequence provides peptides with thermodynamically similar unfolded states. Therefore, the unfolded state can be used as a universal reference state in comparing the thermodynamic properties of the folded coiled-coil structure of the peptides, while varying the configuration of ionized groups, that is, changing the types and number of potential electrostatic interactions. The relative stabilities of these states were determined by monitoring the temperature-induced folding/unfolding of the peptides in solutions with different pH and ionic strength by circular dichroism spectroscopy and scanning microcalorimetry. It was found that, in solutions with low ionic strength, ionic pairs contribute significantly to the stability of the coiled-coil conformation. The stability increases with an increase in the number of ionized groups in the peptide upon changing pH from acidic to neutral. In contrast, in the solutions with high ionic strength, the coiled-coil becomes less stable at neutral pH than at acidic pH. Most surprisingly, the increase in Gibbs energy of stabilization of the coiled-coil state with increasing pH at low ionic strength proceeds with a decrease in the enthalpy and entropy of unfolding. This observation can be explained only by hydration of ionized groups upon unfolding of coiled-coils which is associated with significant negative enthalpy and entropy effects.
The orientation of alpha-helical chains in two-stranded coiled-coils has been shown to be determined by the presence of favorable interchain electrostatic interactions. In this study, we used de novo designed 35-residue peptides to show that when interchain electrostatic interactions are not a factor in coiled-coil formation, the relative positions of Ala residues in the middle heptad can control the parallel or antiparallel orientation of alpha-helical chains in coiled-coils. The peptides formed four-stranded coiled-coils where the helices are either all-parallel or all-antiparallel with respect to their nearest neighbor. The common structural element in these four-stranded coiled-coils is an alternating pair of Ala and Leu residues (Ala-Leu-Ala-Leu) in each of the two planes in the middle heptad. These results indicate that both the relative positions of the Ala residues in the hydrophobic core and the interchain electrostatic interactions between charged residues in the e and g positions should be considered in designing coiled-coils with the desired number of strands in the multiple-stranded assembly. These design elements are also important in orienting functional groups or domains attached to the terminals ends of a coiled-coil carrier.
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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.
A 4.3-kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed-phase HPLC. By the criteria of mass, amino acid composition, and N-terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11-amino acid repeats, one more than the major serum AFP components. The larger protein is completely alpha-helical at 0 degree C, with a melting temperature of 18 degrees C. It is considerably more active as an antifreeze than the three-repeat winter flounder AFP and the four-repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four-repeat winter flounder AFP, including its larger size, additional ice-binding residues, and differences in ice-binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.
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Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt-P. aeruginosa) and C. albicans whole cells (Bt-C. albicans). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.
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.
The objective of this study was to investigate the positional effect of hydrophobic interactions in the alpha-helical interface in controlling the formation of two-stranded and four-stranded coiled-coils. Two disulfide-bridged antiparallel coiled-coils were designed which differ only in the position of a single Ala residue in the middle heptad: in peptide 2H the Ala residues are in register (in the same rung), while in peptide 4H they are not. Data from size-exclusion chromatography and sedimentation equilibrium experiments showed that under benign conditions peptides 2H and 4H were two-stranded and four-stranded coiled-coils respectively. These results, in conjunction with molecular modeling studies, suggests that when four Ala residues are in the same plane of a potential four-stranded coiled-coil, the small side chains of Ala would create a large cavity in the hydrophobic interface of the potential four-stranded structure which is destabilizing and favors the two-stranded, disulfide-bridged coiled-coil. In contrast, an alternating Leu-Ala hydrophobic packing in the two planes distributes the potential cavity over a larger region, which may be partially filled by minor adjustments of the neighboring Leu side chains. As a result, there is still sufficient hydrophobic contact to maintain the four stranded structure.
The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [McInnes, C., et al. (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al. (1995) Biochemistry 34, 16255-16268] reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.
Four linear and four cyclic analogs of gramicidin S (GS) in which D-Phe was replaced with either D-His, D-Ser, D-Tyr or D-Asn have been prepared by solid-phase peptide synthesis and characterized with respect to antibacterial, antifungal and hemolytic activity. Unlike previous reports, GS and a number of cyclic analogs were found to be active against gram-positive as well as gram-negative bacteria. GS showed MICs ranging from 3 to 12.5 micrograms/mL for gram-negative bacteria, compared to MICs of 3 micrograms/mL for gram-positive bacteria. Furthermore, these analogs were also found to exhibit antifungal activity. Unlike the cyclic analogs, all linear analogs were found to be inactive against a wide range of microorganisms tested, and showed low levels of hemolytic activity. The antibacterial activity was found to be highly dependent on the type of assay used, with solution-based assays showing greater activity against gram-negative bacteria than agar-based assays. The GS cyclic analogs were all less toxic than GS itself, with the analog containing the D-Phe to D-Tyr substitution showing the greatest activity of the synthetic analogs. Hemolytic activity in solution against human and sheep red blood cells paralleled antibiotic activity, with those peptides exhibiting greater antibiotic activity generally showing greater hemolytic activity. Membrane destabilization as monitored using the hydrophobic probe N-phenyl-1-naphthylamine was also found to parallel antibacterial and hemolytic activity of cyclic and linear analogs. These results indicate that GS and certain related analogs may have applications as broad-spectrum antibiotics and should be reevaluated for such purposes.
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.