Effects of side chains in helix nucleation differ from helix propagation.

Helix-coil transition theory connects observable properties of the α-helix to an ensemble of microstates and provides a foundation for analyzing secondary structure formation in proteins. Classical models account for cooperative helix formation in terms of an energetically demanding nucleation event (described by the σ constant) followed by a more facile propagation reaction, with corresponding s constants that are sequence dependent. Extensive studies of folding and unfolding in model peptides have led to the determination of the propagation constants for amino acids. However, the role of individual side chains in helix nucleation has not been separately accessible, so the σ constant is treated as independent of sequence. We describe here a synthetic model that allows the assessment of the role of individual amino acids in helix nucleation. Studies with this model lead to the surprising conclusion that widely accepted scales of helical propensity are not predictive of helix nucleation. Residues known to be helix stabilizers or breakers in propagation have only a tenuous relationship to residues that favor or disfavor helix nucleation.

Reversible α-helix formation controlled by a hydrogen bond surrogate.

Strategically placed covalent linkages have been shown to stabilize helical conformations in short peptide sequences. Here we report the synthesis of a stabilized α-helix that utilizes an internal disulfide linkage. Structural analysis indicates that the dynamic nature of the disulfide bridge allows for the reversible formation of an α-helix through oxidation and reduction reactions.

End effects influence short model peptide conformation.

Previously, we derived a P(II) propensity scale using N- and C-terminally blocked host-guest peptide model AcGGXGGNH(2) (X ≠ Gly) and concluded that P(II) represents a dominant conformation in the majority of this series of 19 peptides (Shi et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17964-17968). Recently, Schweitzer-Stenner and co-workers examined a series of eight short host-guest tripeptides with the sequence GXG (X = A, V, F, S, E, L, M, and K) in which both N- and C-ends were unblocked and reported major differences in P(II) content for F, V, and S compared to our scale (Hagarman et al. J. Am. Chem. Soc. 2010, 132, 540-551). We have investigated four representative amino acids (X = A, V, F, and S) in three series of peptides (GXG, AcGXGNH(2), and AcGGXGGNH(2)) as a function of pH in this study. Our data show that P(II) content in the GXG series (X = A, V, F, and S) is pH-dependent and that the conformations of each amino acid differ markedly between the GXG and AcGXGNH(2)/AcGGXGGNH(2) series. Our results indicate that P(II) scales are sequence and context dependent and the presence of proximal charged end groups exerts a strong effect on P(II) population in short model peptides.

Control of bacterial persister cells by Trp/Arg-containing antimicrobial peptides.

Persister cells are dormant phenotypic variants inherent in a bacterial population. They play important roles in chronic infections and present great challenges to therapy due to extremely enhanced tolerance to antibiotics compared to that of normal cells of the same genotype. In this study, we report that cationic membrane-penetrating peptides containing various numbers of arginine and tryptophan repeats are effective in killing persister cells of Escherichia coli HM22, a hyper-persister producer. The activities of three linear peptides [(RW)(n)-NH(2), where n is 2, 3, or 4] and a dendrimeric peptide, (RW)(4D), in killing bacterial persisters were compared. Although the dendrimeric peptide (RW)(4D) requires a lower threshold to kill planktonic persisters, octameric peptide (RW)(4)-NH(2) is the most effective against planktonic persister cells at high concentrations. For example, treatment with 80 µM (RW)(4)-NH(2) for 60 min led to a 99.7% reduction in the number of viable persister cells. The viability of persister cells residing in surface-attached biofilms was also significantly reduced by (RW)(4)-NH(2) and (RW)(4D). These two peptides were also found to significantly enhance the susceptibility of biofilm cells to ofloxacin. The potency of (RW)(4)-NH(2) was further marked by its ability to disperse and kill preformed biofilms harboring high percentages of persister cells. Interestingly, approximately 70% of the dispersed cells were found to have lost their intrinsic tolerance and become susceptible to ampicillin if not killed directly by this peptide. These results are helpful for better understanding the activities of these peptides and may aid in future development of more effective therapies of chronic infections.

Effects of Trp- and Arg-containing antimicrobial-peptide structure on inhibition of Escherichia coli planktonic growth and biofilm formation.

Biofilms are sessile microbial communities that cause serious chronic infections with high morbidity and mortality. In order to develop more effective approaches for biofilm control, a series of linear cationic antimicrobial peptides (AMPs) with various arginine (Arg or R) and tryptophan (Trp or W) repeats [(RW)(n)-NH(2), where n = 2, 3, or 4] were rigorously compared to correlate their structures with antimicrobial activities affecting the planktonic growth and biofilm formation of Escherichia coli. The chain length of AMPs appears to be important for inhibition of bacterial planktonic growth, since the hexameric and octameric peptides significantly inhibited E. coli growth, while tetrameric peptide did not cause noticeable inhibition. In addition, all AMPs except the tetrameric peptide significantly reduced E. coli biofilm surface coverage and the viability of biofilm cells, when added at inoculation. In addition to inhibition of biofilm formation, significant killing of biofilm cells was observed after a 3-hour treatment of preformed biofilms with hexameric peptide. Interestingly, treatment with the octameric peptide caused significant biofilm dispersion without apparent killing of biofilm cells that remained on the surface; e.g., the surface coverage was reduced by 91.5 + or - 3.5% by 200 microM octameric peptide. The detached biofilm cells, however, were effectively killed by this peptide. Overall, these results suggest that hexameric and octameric peptides are potent inhibitors of both bacterial planktonic growth and biofilm formation, while the octameric peptide can also disperse existing biofilms and kill the detached cells. These results are helpful for designing novel biofilm inhibitors and developing more effective therapeutic methods.

Antimicrobial dendrimer active against Escherichia coli biofilms.

We have investigated the ability of a previously reported antimicrobial peptide dendrimer (RW)(4D) to inactivate Escherichia coli RP437 in planktonic culture and in biofilms. The results show that the dendrimer inhibits bacterial growth in both planktonic and biofilm states. Live/Dead staining assays reveal that most bacteria in a preformed biofilm lose viability after treatment with this peptide. This result is in marked contrast to most existing reports that antimicrobial peptides are ineffective against mature bacterial biofilms.

Interactions of recombinant HMGB proteins with branched RNA substrates.

The high mobility group protein HMGB1 is a highly abundant chromosomal protein known to interact preferentially with DNA that is branched, bent or otherwise structurally altered. Biologically the protein is thought to facilitate promoter attachment by transcription factors. Recently, however, HMGB1 has been shown to have biological roles beyond that of an architectural DNA-binding protein. Here we investigate the binding interactions of recombinant HMGB1 proteins with two branched RNA's E. coli 5S rRNA and the group I intron ribozyme from Azoarcus pre-tRNA(Ile). Using competitive electrophoretic mobility and circular dichroism binding assays, we show that HMGB proteins bind both substrates with high affinity. We also report that a recombinant rat HMGB protein, rHMGB1b, inhibits RNA cleavage by the ribozyme. These results raise the possibility that HMGB proteins possess structure dependent RNA binding activity and can modulate RNA processing as well as transcription.

A heterospecific leucine zipper tetramer.

Protein-protein interactions play an essential role in the assembly of the macromolecular complexes that form functional networks and control cellular behavior. Elucidating principles of molecular recognition governing potentially complex interfaces is a challenging goal for structural and systems biology. Extensive studies of alpha-helical coiled coils have provided fundamental insight into the determinants of one seemingly tractable class of oligomeric protein interfaces. We report here that two different valine-containing mutants of the GCN4 leucine zipper that fold individually as four-stranded coiled coils associate preferentially in mixtures to form an antiparallel, heterotetrameric structure. X-ray crystallographic analysis reveals that the coinciding hydrophobic interfaces of the hetero- and homotetramers differ in detail, thereby controlling their partnering and structural specificity. Equilibrium disulfide exchange and thermal denaturation experiments show that the 50-fold preference for heterospecificity is determined by interfacial van der Waals interactions and hydrophobicity. Parallel studies of two alanine-containing variants confirm the above-mentioned interpretation of the basis and mechanism of this heterospecificity. Our results suggest that coiled-coil recognition is an inherently geometric process in which heterotypic interaction specificity derives from a complementarity of both shape and chemistry.

Trapping a folding intermediate of the alpha-helix: stabilization of the pi-helix.

We report the design, synthesis, and characterization of a short peptide trapped in a pi-helix configuration. This high-energy conformation was nucleated by a preorganized pi-turn, which was obtained by replacing an N-terminal intramolecular main chain i and i + 5 hydrogen bond with a carbon-carbon bond. Our studies highlight the nucleation parameter as a key factor contributing to the relative instability of the pi-helix and allow us to estimate fundamental helix-coil transition parameters for this conformation.

Synthesis and biological evaluation of novel 1,3,5-triazine derivatives as antimicrobial agents.

Numerous studies have contributed to the development of natural and synthetic antimicrobial peptides as a prospective source of antibiotic agents. Based on the concept that cationic charge, bulk, and lipophilicity are major factors determining antibacterial activity in these peptides, we designed and screened several combinatorial libraries based on 1,3,5-triazine as a template. A set of compounds were identified to show potent antimicrobial activity together with low hemolytic activity.

Conformational specificity of the lac repressor coiled-coil tetramerization domain.

Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of alpha-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 A reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.

Length effects in antimicrobial peptides of the (RW)n series.

A class of antimicrobial peptides involved in host defense consists of sequences rich in Arg and Trp-R and -W. Analysis of the pharmacophore in these peptides revealed that chains as short as trimers of sequences such as WRW and RWR have antimicrobial activity (M. B. Strom, B. E. Haug, M. L. Skar, W. Stensen, T. Stiberg, and J. S. Svendsen, J. Med. Chem. 46:1567-1570, 2003). To evaluate the effect of chain length on antimicrobial activity, we synthesized a series of peptides containing simple sequence repeats, (RW)n-NH2 (where n equals 1, 2, 3, 4, or 5), and determined their antimicrobial and hemolytic activity. The antimicrobial activity of the peptides increases with chain length, as does the hemolysis of red blood cells. Within the experimental error, longer peptides (n equals 3, 4, or 5) show similar values for the ratio of hemolytic activity to antibacterial activity, or the hemolytic index. The (RW)3 represents the optimal chain length in terms of the efficacy of synthesis and selectivity as evaluated by the hemolytic index. Circular dichroism spectroscopy indicates that these short peptides appear to be unfolded in aqueous solution but acquire structure in the presence of phospholipids. Interaction of the peptides with model lipid vesicles was examined using tryptophan fluorescence. The (RW)n peptides preferentially interact with bilayers containing the negatively charged headgroup phosphatidylglycerol relative to those containing a zwitterionic headgroup, phosphatidylcholine.

Self-assembly of coiled-coil tetramers in the 1.40 A structure of a leucine-zipper mutant.

The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.

A parallel coiled-coil tetramer with offset helices.

Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between alpha helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of alpha-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete alpha-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 A resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.

A seven-helix coiled coil.

Coiled-coil proteins contain a characteristic seven-residue sequence repeat whose positions are designated a to g. The interacting surface between alpha-helices in a classical coiled coil is formed by interspersing nonpolar side chains at the a and d positions with hydrophilic residues at the flanking e and g positions. To explore how the chemical nature of these core amino acids dictates the overall coiled-coil architecture, we replaced all eight e and g residues in the GCN4 leucine zipper with nonpolar alanine side chains. Surprisingly, the alanine-containing mutant forms a stable alpha-helical heptamer in aqueous solution. The 1.25-A resolution crystal structure of the heptamer reveals a parallel seven-stranded coiled coil enclosing a large tubular channel with an unusual heptad register shift between adjacent staggered helices. The overall geometry comprises two interleaved hydrophobic helical screws of interacting cross-sectional a and d layers that have not been seen before. Moreover, asparagines at the a positions play an essential role in heptamer formation by participating in a set of buried interhelix hydrogen bonds. These results demonstrate that heptad repeats containing four hydrophobic positions can direct assembly of complex, higher-order coiled-coil structures with rich diversity for close packing of alpha-helices.

Conformational transition between four and five-stranded phenylalanine zippers determined by a local packing interaction.

Alpha-helical coiled coils play a crucial role in mediating specific protein-protein interactions. However, the rules and mechanisms that govern helix-helix association in coiled coils remain incompletely understood. Here we have engineered a seven heptad "Phe-zipper" protein (Phe-14) with phenylalanine residues at all 14 hydrophobic a and d positions, and generated a further variant (Phe-14(M)) in which a single core Phe residue is substituted with Met. Phe-14 forms a discrete alpha-helical pentamer in aqueous solution, while Phe-14(M) folds into a tetrameric helical structure. X-ray crystal structures reveal that in both the tetramer and the pentamer the a and d side-chains interlock in a classical knobs-into-holes packing to produce parallel coiled-coil structures enclosing large tubular cavities. However, the presence of the Met residue in the apolar interface of the tetramer markedly alters its local coiled-coil conformation and superhelical geometry. Thus, short-range interactions involving the Met side-chain serve to preferentially select for tetramer formation, either by inhibiting a nucleation step essential for pentamer folding or by abrogating an intermediate required to form the pentamer. Although specific trigger sequences have not been clearly identified in dimeric coiled coils, higher-order coiled coils, as well as other oligomeric multi-protein complexes, may require such sequences to nucleate and direct their assembly.