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Neville R Kallenbach

Publications and source records attributed to Neville R Kallenbach.

At least 19 recordsLinked to original sources
PubMed2006

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.

Crystallography, X-Ray↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

Multivalent antimicrobial peptides from a reactive polymer scaffold.

We report an application of the principle of multivalency to create new antimicrobial agents using the reactive polymaleic anhydride (PMA) chain to link antimicrobial tetrapeptides to afford multivalent variants containing approximately 40 monomer units. Relative to the free peptides, the product shows a 10-fold improvement in IC(50) without provoking more severe hemolysis of red blood cells. Thus, multivalency or polyvalency may offer a route to enhance the activity of antimicrobial peptides.

Anti-Infective Agents↗
PubMed2006

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.

Amino Acid Motifs↗
PubMed2006

PII structure in the model peptides for unfolded proteins: studies on ubiquitin fragments and several alanine-rich peptides containing QQQ, SSS, FFF, and VVV.

A great deal of attention has been paid lately to the structures in unfolded proteins due to the recent discovery of many biologically functional but natively unfolded proteins and the far-reaching implications of order in unfolded states for protein folding. Recently, studies on oligo-Ala, oligo-Lys, oligo-Asp, and oligo-Glu, as well as oligo-Pro, have indicated that the left-handed polyproline II (PII) is the major local structure in these short peptides. Here, we show by NMR and CD studies that ubiquitin fragments, model unfolded peptides composed of nonrepeating amino acids, and four alanine-rich peptides containing QQQ, SSS, FFF, and VVV sequences are all present in aqueous solution predominantly in the extended PII or beta conformation. The results from this and related studies indicate that PII might be a major backbone conformation in unfolded proteins. The presence of defined local backbone structure in unfolded proteins is inconsistent with predictions from random coil models.

Alanine↗
PubMed2006

Antiparallel four-stranded coiled coil specified by a 3-3-1 hydrophobic heptad repeat.

Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

Basic-Leucine Zipper Transcription Factors↗
PubMed2005

Polyproline II propensities from GGXGG peptides reveal an anticorrelation with beta-sheet scales.

There is growing appreciation of the functional relevance of unfolded proteins in biology. However, unfolded states of proteins have proven inaccessible to the usual techniques for high-resolution structural and energetic characterization. Unfolded states are still generally conceived of as statistical coils, based on the pioneering work of Flory [(1969) Statistical Mechanics of Chain Molecules (Wiley, New York)] and Tanford [(1968) Adv. Protein Chem. 23, 121-282]. Recently, several lines of independent evidence have raised doubts about the random coil model and offer support for alternative views. Here, we show that polyproline II conformation is dominant in a host-guest peptide model AcGGXGGNH(2) (X not equal glycine), in equilibrium predominantly with beta-structure. This result is inconsistent with a random coil model and the general view that these peptides are unstructured. By calculating a set of apparent DeltaG values from the measured coupling constants of the backbone amides, we can construct a polyproline II scale that correlates negatively with beta-sheet scales.

Magnetic Resonance Spectroscopy↗
PubMed2005

Neighbor effect on PPII conformation in alanine peptides.

The polyproline II (PPII) conformation is dominant in short alanine oligomers. The noncooperativity of PPII structure in alanine peptides indicates that PPII in water is locally determined and that alanine neighbors are consistent with Flory's isolated pair hypothesis. However, neighbor effects from beta-branched or bulky aromatic residues tend to increase the Phi angle of the nearest neighbor as observed in coil library data. Here we demonstrate directly the neighbor effect using short alanine model peptides GGAAAGG, GGLnALnGG (Ln is norleucine), GGIAAGG, and GGIAIGG. The far-UV CD spectra, NMR 3JalphaN coupling constant, and H-D hydrogen exchange measurements reveal that Ile reduces the PPII content of the probe Ala side chain relative to Ala or norLeu. The free energy differences are consistent with predictions from electrostatic solvation free energy (ESF) calculations. The results indicate that prediction of PPII propensities or scales requires including the neighbor effect.

Alanine↗
PubMed2004

Solvent dependence of PII conformation in model alanine peptides.

Alanine residues in two model peptides, the pentapeptide AcGGAGGNH(2) and the 11mer AcO(2)A(7)O(2)NH(2), have been reported to have substantial PII conformation in water. The PII structure in both peptides is sensitive to solvent. In the presence of the organic solvent TFE, the conformation of the pentamer changes from PII to internally H-bonded gamma or beta turns, while the chain with seven alanines forms alpha helix. The PII structure in the 11mer is more stable than that in the shorter peptide as the TFE concentration increases. For the pentamer, a comparison of short-chain aliphatic alcohols to water shows that the PII content decreases in the order water > methanol > ethanol > 2-propanol, linearly according to empirical scales of solvent polarity. Thus, depending on the extent of local solvation as folding progresses, the peptide backbone as modeled by alanine oligomers shifts from PII to internally H-bonded (gamma or beta turn) conformations and to alpha helix in longer segments. On the other hand, the PII content of AcO(2)A(7)O(2)NH(2) increases significantly in the presence of guanidine, as does that of oligoproline peptides, while detergent sodium dodecyl sulfate (SDS) favors alpha helix in this peptide. The shorter peptide does not show a parallel increase in PII with guanidine.

Circular Dichroism↗
PubMed2004

Atomic structure of a tryptophan-zipper pentamer.

Coiled-coil motifs are ubiquitous mediators of specific protein-protein interactions through the formation of interlocking hydrophobic seams between alpha-helical chains. Residues that form these seams occur at the first (a) and fourth (d) positions of a characteristic 7-aa repeat and are primarily aliphatic. The potential of aromatic residues to promote helix association in a coiled coil was explored by engineering a "Trp-zipper" protein with Trp residues at all 14 a and d positions. The protein forms a discrete, stable, alpha-helical pentamer in water at physiological pH. Its 1.45-A crystal structure reveals a parallel, five-stranded coiled coil, a previously uncharacterized type of "knobs-into-holes" packing interaction between interfacial Trp side chains, and an unusual approximately 8-A-diameter axial channel lined with indole rings that is filled with polyethylene glycol 400 and water and sulfate ion molecules. The engineered Trp-zipper pentamer enlarges current views of coiled-coil assembly, molecular recognition, and protein engineering, and may serve as a soluble model for membrane ion channels.

Amino Acid Motifs↗
PubMed2004

The polyproline II conformation in short alanine peptides is noncooperative.

The finding that short alanine peptides possess a high fraction of polyproline II (PII) structure (Phi=-75 degrees, Psi=+145 degrees ) at low temperature has broad implications for unfolded states of proteins. An important question concerns whether or not this structure is locally determined or cooperative. We have monitored the conformation of alanine in a series of model peptides AcGGAnGGNH2 (n=1-3) over a temperature range from -10 degrees C to +80 degrees C. Use of 15N-labeled alanine substitutions makes it possible to measure 3JalphaN coupling constants accurately over the full temperature range. Based on a 1D next-neighbor model, the cooperative parameter sigma of PII nucleation is evaluated from the coupling constant data. The finding that sigma is close to unity (1 +/- 0.2) indicates a noncooperative role for alanine in PII structure formation, consistent with statistical surveys of the Protein Data Bank that suggest that most PII structure occurs in isolated residues. Lack of cooperativity in these models implies that hydration effects that influence PII conformation in water are highly localized. Using a nuclear Overhauser effect ratio strategy to define the alanine Psi angle, we estimate that, at 40 degrees C, the time-averaged alanine conformation (Phi=-80 degrees, Psi=+170 degrees ) deviates from canonical PII structure, indicating that PII melts at high temperature. Thus, the high-temperature state of short alanine peptides seems to be an unfolded ensemble with higher distribution in the extended beta structure basin, but not a coil.

Alanine↗
PubMed2004

Vibrational Raman optical activity characterization of poly(l-proline) II helix in alanine oligopeptides.

A vibrational Raman optical activity (ROA) study of a series of alanine peptides in aqueous solution is presented. The seven-alanine peptide Acetyl-OOAAAAAAAOO-Amide (OAO), recently shown by NMR and UVCD to adopt a predominantly poly(l-proline II) (PPII) helical conformation in aqueous solution, gave an ROA spectrum very similar to that of disordered poly(l-glutamic acid) which has long been considered to adopt the PPII conformation, both being dominated by a strong positive extended amide III ROA band at approximately 1319 cm-1 together with weak positive amide I ROA intensity at approximately 1675 cm-1. A series of alanine peptides Ala2-Ala6 studied in their cationic states in aqueous solution at low pH displayed ROA spectra which steadily evolved toward that of OAO with increasing chain length. As well as confirming that alanine peptides can support the PPII conformation in aqueous solution, our results also confirm the previous ROA band assignments for PPII structure, thereby reinforcing the foundation for ongoing ROA studies of unfolded and partially folded proteins.

Amides↗
PubMed2004

Helix formation and the unfolded state of a 52-residue helical protein.

A growing class of proteins in biological processes has been found to be unfolded on isolation under normal solution conditions. We have used NMR spectroscopy to characterize the structural and dynamic properties of the unfolded and partially folded states of a 52-residue alanine-rich protein (Ala-14) at temperatures from -5 degrees C to 40 degrees C. At 40 degrees C, alanine residues in Ala-14 adopt phi and psi angles, consistent with a significant ensemble population of polyproline II conformation. Analysis of relaxation rates in the protein reveals that a series of residues, Gln 35-Ala 36-Ala 37-Lys 38-Asp 39-Asp 40-Ala 41-Ala 42, displays slow motional dynamics at both -5 degrees C and 40 degrees C. Temperature-dependent chemical shift changes indicate that this region is the site of helix initiation. The remaining N-terminal residues become increasingly dynamic as they extend from the nucleation site. The C terminus remains dynamic and changes less with temperature, indicating it is relatively unstructured. Ala-14 provides a high-resolution portrait of the unfolded state and the process of helix nucleation and propagation in the absence of tertiary contacts, information that bears on early events in protein folding.

Alanine↗
PubMed2003

The pentapeptide GGAGG has PII conformation.

Most of what we know about proteins reflects their native folded structure. Much less is understood about the structure of unfolded proteins, which tends to be referred to as "random coil", lacking extended alpha-helix or beta-strand structure. Recent work suggests that unfolded proteins might adopt significant population of PII structure, an extended left-handed helix found in collagen and proline-rich peptides. A series of short peptides AcGGXGGNH2 has been adopted as a model for studying unfolded protein structure because of the minimal steric effect imposed by flanking glycines. Peptide AcGGAGGNH2 makes possible a host-guest conformation analysis of the middle residue alanine. NMR experiments reveal that the Phi and Psi dihedral angles of the central alanine are -73 degrees and 125 degrees , respectively, placing the alanine in the PII region of the Ramachandran plot. Circular dichroism shows a typical PII spectrum with a strong negative absorbance at 190 nm. Temperature experiments show the alanine structure shifts to increasing beta-strand at high temperature. Because the alanine side chain most closely represents unsubstituted peptide backbone, these results have significant implications for the conformational entropy of unfolded polypeptide chains.

Alanine↗
PubMed2002

Non-classical helix-stabilizing interactions: C-H...O H-bonding between Phe and Glu side chains in alpha-helical peptides.

The classical picture of H-bonds has evolved considerably. In contrast to earlier expectations, C-H...O H-bonds are now known to be prevalent in both small organic and large biological systems. However, there are few reports on the energetic contribution of C-H...O H-bonds in protein or polypeptide systems and we do not know whether such interactions are stabilizing. Here we investigate C-H...O H-bonding interactions between Phe and Glu side chains by determining their effects on the helicity of model alpha-helical peptides using a combination of CD and NMR spectroscopy. The results suggest that Glu/Phe C-H...O H-bonding interactions stabilize helical structure, but only in the orientation Glu --> Phe (N --> C). Each Glu --> Phe (N --> C) interaction can contribute approximately -0.5 kcal mol(-1) to the stability of helical peptide. In the reverse orientation, Phe --> Glu (N --> C) appears to contribute negligibly. pH titrations provide further evidence for the existence of C-H...O H-bonds. The C-H...O H-bonding interactions in these peptides are insensitive to the screening effect of added neutral salt. Our results provide quantitative energetic information on C-H...O H-bonds that should be useful for empirical force-field calibration.

Amino Acid Sequence↗