Nonpolar contributions to conformational specificity in assemblies of designed short helical peptides

A series of designed short helical peptides was used to study the effect of nonpolar interactions on conformational specificity. The consensus sequence was designed to obtain short helices (17 residues) and to minimize the presence of interhelical polar interactions. Furthermore, the sequence contained a heptad repeat (abcdefg), where positions a and d were occupied by hydrophobic residues Leu, Ile, or Val, and positions e and g were occupied by Ala. The peptides were named according to the identities of the residues in the adeg positions, respectively. The peptides llaa, liaa, ilaa, iiaa, ivaa, viaa, lvaa, vlaa, and vvaa were synthesized, and their characterization revealed marked differences in specificity. An experimental methodology was developed to study the nine peptides and their pairwise mixtures. These peptides and their mixtures formed a vast array of structural states, which may be classified as follows: helical tetramers and pentamers, soluble and insoluble helical aggregates, insoluble unstructured aggregates, and soluble unstructured monomers. The peptide liaa formed stable helical pentamers, and iiaa and vlaa formed stable helical tetramers. Disulfide cross-linking experiments indicated the presence of an antiparallel helix alignment in the helical pentamers and tetramers. Rates of amide proton exchange of the tetrameric form of vlaa were 10-fold slower than the calculated exchange rate for unfolded vlaa. In other work, the control of specificity has been attributed to polar interactions, especially buried polar interactions; this work demonstrated that subtle changes in the configuration of nonpolar interactions resulted in a large variation in the extent of conformational specificity of assemblies of designed short helical peptides. Thus, nonpolar interactions can have a significant effect on the conformational specificity of oligomeric short helices.     

Engineering of the hydrophobic core of an alpha-helical coiled coil

The amino acid sequence that forms the alpha-helical coiled coil structure has a representative heptad repeat denoted by defgabc, according to their positions. Although the a and d positions are usually occupied by hydrophobic residues, hydrophilic residues at these positions sometimes play important roles in natural proteins. We have manipulated a few amino acids at the a and d positions of a de novo designed trimeric coiled coil to confer new functions to the peptides. The IZ peptide, which has four heptad repeats and forms a parallel triple-stranded coiled coil, has Ile at all of the a and d positions. We show three examples: (1) the substitution of one Ile at either the a or d position with Glu caused the peptide to become pH sensitive; (2) the metal ion induced alpha-helical bundles were formed by substitutions with two His residues at the d and a positions for a medium metal ion, and with one Cys residue at the a position for a soft metal ion; and (3) the AAB-type heterotrimeric alpha-helical bundle formation was accomplished by a combination of Ala and Trp residues at the a positions of different peptide chains. Furthermore, we applied these procedures to prepare an ABC-type heterotrimeric alpha-helical bundle and a metal ion-induced heterotrimeric alpha-helical bundle.     

Conformation specificity and arene binding in a peptide composed only of Lys, Ile, Ala and Gly

The first life on Earth is believed to have been based on RNA, but might have taken advantage of amino acids and short peptides which form readily under conditions like those of the primitive Earth. We have shown that simple peptides adopt specifically folded four-helix bundle structures that can recognize and cleave RNA. Here, to explore the limits of conformational specificity, we characterize a simpler peptide composed of just Lys, Ile, Ala, and Gly called KIA7I. Using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations, we find kinks in the helices of KIA7I and multiple C-terminal conformations. These results suggest that the C-terminal Ile residue does not completely occupy the hydrophobic pocket that is filled by aromatic side-chains in well-folded KIA7 variants. The capacity of arenes to fill this cavity was tested. Using NMR, we show that benzene and phenol can bind KIA7I, but do not bind the well-folded variant KIA7W or hen egg white lysozyme. Benzene also binds Aβ_1–40, a mostly disordered polypeptide implicated in Alzheimer’s disease. 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence is further enhanced in the presence of both KIA7I and arenes relative to KIA7I alone. This ANS fluorescence enhancement is stronger for smaller and less polar arenes and less ordered KIA variants. These results suggest that arenes are not confined to the pocket, but penetrate and loosen the hydrophobic core of KIA7I.     

Reversible assembly of helical filaments by de novo designed minimalist peptides

We have designed a series of 15 short, helical de novo peptides consisting of lysine, isoleucine, and alanine. We have termed this the KIA series. These peptides differ only in their hydrophobic interface, and thus their self-association is largely a consequence of hydrophobic interactions. One of these peptides, KIA13, forms insoluble helical fibers at specific NaCl concentrations. We have used CD spectroscopy, turbidity assays, and in situ tapping mode atomic force microscopy to characterize the reversible assembly pathway for this peptide. It is unfolded at low NaCl concentration, and forms helical, soluble fibers resembling a coiled-coil conformation at intermediate NaCl concentrations, and rope-like insoluble fibers at high NaCl concentrations. Reducing the NaCl concentration completely reverses this process. Another peptide from the KIA series specifically inhibits the formation of the insoluble KIA13 fibers, and reverses the process to some extent. This work sheds light onto protein fibrillogenesis and offers intriguing possibilities for the use of these types of peptides in drug delivery and biomaterials applications.     

The formation of straight and twisted filaments from short tau peptides

We studied fibril formation in a family of peptides based on PHF6 (VQIVYK), a short peptide segment found in the microtubule binding region of tau protein. N-Acetylated peptides AcVYK-amide (AcVYK), AcIVYK-amide (AcPHF4), AcQIVYK-amide (AcPHF5), and AcV-QIVYK-amide (AcPHF6) rapidly formed straight filaments in the presence of 0.15 m NaCl, each composed of two laterally aligned protofilaments approximately 5 nm in width. X-ray fiber diffraction showed the omnipresent sharp 4.7-A reflection indicating that the scattering objects are likely elongated along the hydrogen-bonding direction in a cross-beta conformation, and Fourier transform IR suggested the peptide chains were in a parallel (AcVYK, AcPHF6) or antiparallel (AcPHF4, AcPHF5) beta-sheet configuration. The dipeptide N-acetyl-YK-amide (AcYK) formed globular structures approximately 200 nm to 1 microm in diameter. The polymerization rate, as measured by thioflavin S binding, increased with the length of the peptide going from AcYK --> AcPHF6, and peptides that aggregated most rapidly displayed CD spectra consistent with beta-sheet structure. There was a 3-fold decrease in rate when Val was substituted for Ile or Gln, nearly a 10-fold decrease when Ala was substituted for Tyr, and an increase in polymerization rate when Glu was substituted for Lys. Twisted filaments, composed of four laterally aligned protofilaments (9-19 nm width, approximately 90 nm half-periodicity), were formed by mixing AcPHF6 with AcVYK. Taken together these results suggest that the core of PHF6 is localized at VYK, and the interaction between small amphiphilic segments of tau may initiate nucleation and lead to filaments displaying paired helical filament morphology.     

Role of N-t-Boc group in helix initiation in a novel tetrapeptide.

Protecting groups in N- and C-terminal positions play a decisive role in the conformational preference of smaller peptides. Conformational analysis of tetrapeptide derivatives containing Ala, Ile and Gly residues was performed. Peptide 1, Boc-Ala-Ile-Ile-Gly-OMe (Boc: tert-butyloxycarbonyl) has a predominantly helical turn conformation in all the alcoholic solvents studied, whereas in the solid state it has a beta-sheet conformation. In contrast, peptide 2, Ac-Ala-Ile-Ile-Gly-OMe (Ac: acetyl) has a random coil conformation in solution. The FTIR spectrum of peptide 1 shows a lower frequency of urethane carbonyl, indicating involvement of the carbonyl group in hydrogen bonding in the helical turn.     

Soft metal ions, Cd(II) and Hg(II), induce triple-stranded alpha-helical assembly and folding of a de novo designed peptide in their trigonal geometries

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded alpha-helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple-stranded alpha-helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 +/- 0.8 microM for Cd(II). The triple-stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl- ions. The 113Cd NMR resonance was connected with the betaH protons of the cysteine residue by 1H-113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg(II) also induced the assembly of the peptide into a triple-stranded alpha-helical bundle below the Hg(II)/peptide ratio of 1/3. With excess Hg(II), however, the alpha-helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.     

Rotational orientation of monomers within a designed homo-oligomer transmembrane helical bundle

A peptide designed to form a homo-oligomeric transmembrane helical bundle was reconstituted into lipid bilayers and studied by using (2)H NMR (nuclear magnetic resonance) with magic angle spinning to confirm that the helical interface corresponds to the interface intended in the design. The peptide belongs to a family of model peptides derived from a membrane-solubilized version of the water-soluble coiled-coil GCN4-P1. The variant studied here contains two asparagines thought to engage in interhelical hydrogen bonding critical to the formation of a stable trimer. For the NMR studies, three different peptides were synthesized, each with one of three consecutive leucines in the transmembrane region deuterium labeled. Prior to NMR data collection, polarized infrared spectroscopy was used to establish that the peptides were reconstituted in lipid bilayers in a transmembrane helical conformation. The (2)H NMR line shapes of the three different peptides are consistent with a trimer structure formed by the designed peptide that is stabilized by inter-helical hydrogen bonding of asparagines at positions 7 and 14.     

Immunization with synthetic peptides containing a defined malaria epitope induces a highly diverse cytotoxic T lymphocyte response. Evidence that two peptide residues are buried in the MHC molecule.

In the present study, we have explored ways of inducing a CTL response to a previously defined H-2Kd MHC class I restricted epitope in the circumsporozoite (CS) protein of Plasmodium berghei, and studied in detail the fine specificity of the response. We found that the s.c. injection of a variety of synthetic peptides emulsified in Freund's adjuvant efficiently induced a specific CTL response in (BALB/c x C57BL/6)F1 (H-2d x H-2b) mice. In contrast, BALB/c mice responded only marginally, consistent with the possible requirement for a concomitant Th response that would be provided by the C57BL/6 strain. Similar to our previous observations in analyzing CTL clones from sporozoite-immunized mice, the CTL response induced by peptide immunization was in part cross-reactive with an epitope from the Plasmodium yoelii species. The minimal P. berghei CS epitope, the octapeptide PbCS 253-260, was studied in detail by the analysis of a series of variant CS peptides containing single Ala substitutions. The relative antigenic activity for each variant peptide was calculated for 28 different CTL clones. Overall, the response to this P. berghei CTL epitope appeared to be extremely diverse in terms of fine specificity. This was evident among the CTL derived from sporozoite-immunized mice, as well as among those from peptide-immunized animals. The heterogeneity found at the functional level correlates with the highly diverse TCR repertoire that we have found for the same series of CTL clones in a study that is reported separately. The relative competitor activity for each Ala-substituted peptide was also determined in a quantitative functional competition assay. For the residues (Tyr253 and Ile260) within the 8-mer CS peptide, substitution with Ala reduced competitor activity by at least 40-fold, and for two others the reduction was 5- to 10-fold. When the relative antigenic activity for each CTL/peptide combination was normalized to the relative competitor activity of the peptide, a striking pattern emerged. The two residues that most affected competitor activity showed no additional effect on recognition beyond that observed for competition. In marked contrast, Ala substitutions at the other five positions tested varied widely, depending on the CTL/peptide combination. This pattern not only supports a model whereby the Tyr253 and Ile260 residues anchor the peptide to the Kd molecule, but also implies that they are virtually inaccessible to the TCR.     

Design and synthesis of 3alpha-helix peptides forming a cavity for a fluorescent ligand

As a model of receptor protein, a series of 3alpha-helix bundle peptides constructed on a template peptide were designed so as to possess a hydrophobic cavity. The size of cavity was modulated by simple replacements of Leu residues to Ala residues in the hydrophobic core. Binding abilities to 8-anilino-1-naphthalenesulfonic acid (ANS) were estimated by the increase of fluorescence intensity. The peptide having three or four Ala residues in the hydrophobic core remarkably increased the binding ability for ANS, though the peptide having two Ala residues gave an inefficient cavity for ANS. The peptide having six Ala residues decreased the binding ability due to crucial destabilization of the helix bundle structure. This scaffold can be utilized to a receptor model, while further tuning of the sequence is necessary.     

Photo-control of peptide helix content by an azobenzene cross-linker: steric interactions with underlying residues are not critical

Photo-control of protein conformation could prove useful for probing function in diverse biological systems. Recently, we reported photo-switching of helix content in a short peptide containing an azobenzene cross-linker between cysteine residues at positions i and i + 7 in the sequence. In the original sequence, underlying residues at positions i + 3 and i + 4 were made bulky as preliminary modelling suggested that this would enhance photo-control of helix content. To test this hypothesis, peptides with Val, Aib; Ile, Aib; and Ala, Ala at positions i + 3 and i + 4 were synthesized, cross-linked and characterized. Before cross-linking, the peptides show distinct conformational behaviours: two with differing helix/coil mixtures whereas the other has a circular dichroism (CD) spectrum characteristic of beta-sheet and a tendency to aggregate. However, upon cross-linking the peptides have very similar CD spectra: predominantly random coil in the dark but predominantly helical upon irradiation. These results refute the original hypothesis. Steric interactions between the linker and underlying residues do not appear to be critical for photo-switching behaviour. When the cross-linking bridge is lengthened by replacing the i, i + 7 cysteine residues with homocysteine, a lower degree of photo-control of helicity is observed. Furthermore, a non-cross-linking version of the azobenzene reagent is shown not to produce any photo-control of helicity. We conclude that the intramolecular cross-link is essential for photo-switching and that it should be applicable to a wide range of peptides and proteins.     

Dramatic structural and thermodynamic consequences of repacking a protein's hydrophobic core

BACKGROUND: Rop is an RNA binding, dimeric, four-helix bundle protein with a well-defined, regular hydrophobic core ideally suited for redesign studies. A family of Rop variants in which the hydrophobic core was systematically redesigned has previously been created and characterized. RESULTS: We present a structural and thermodynamic analysis of Ala2Ile2-6, a variant of Rop with an extensively redesigned hydrophobic core. The structure of Ala2Ile2-6 reveals a completely new fold formed by a conformational "flip" of the two protomers around the dimeric interface. The free-energy profile of Ala2Ile2-6 is also very different from that of wild-type Rop. Ala2Ile2-6 has a higher melting temperature than Rop, but undergoes a slightly smaller free-energy change on unfolding. CONCLUSIONS: The structure of Ala2Ile2-6, along with molecular modeling results, demonstrate the importance of tight packing of core residues and the adoption of favorable core side chain rotamer values in determining helix-helix interactions in the four-helix bundle fold. Structural disorder at the N and C termini of Ala2Ile2-6 provides a basis for the large differences in the enthalpy and entropy of Ala2Ile2-6 folding compared with wildtype Rop.     

The effect of hydrophobic analogues of the type I winter flounder antifreeze protein on lipid bilayers

The effect of four synthetic analogues of the 37-residue winter flounder type I antifreeze protein (AFP), which contain four Val, Ala or Ile residues in place of Thr residues at positions 2, 13, 24 and 37 and two additional salt bridges, on the binary lipid system prepared from a 1:1 mixture of the highly unsaturated DGDG and saturated DMPC has been determined using FTIR spectroscopy. In contrast to the natural protein, which increases the thermotropic phase transition, the Thr, Val and Ala analogues decreased the thermotropic phase transitions of the liposomes by 2.2 degrees Celsius, 3.4 degrees Celsius and 2.4 degrees Celsius, while the Ile analogue had no effect on the transition. Experiments performed using perdeuterated DMPC showed that the Ala and Thr peptides interacted preferentially with the DGDG in the lipid mixture, while the Val peptide showed no preference for either lipid. The results are consistent with interactions involving the hydrophobic face of type I AFPs and model bilayers, i.e. the same face of the protein that is responsible for antifreeze properties. The different effects correlate with the helicity of the peptides and suggest that the solution conformation of the peptides has a significant role in determining the effects of the peptides on thermotropic membrane phase transitions.     

Dissecting quasi-equivalence in nonenveloped viruses: membrane disruption is promoted by lytic peptides released from subunit pentamers, not hexamers

Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question with Nudaurelia capensis omega virus (NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiated in vitro and is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.     

Cleavage site specificity of vertebrate collagenases

A series of synthetic peptide substrates for vertebrate collagenase having the structure Ac-Pro-Leu-Gly-X-Leu-Gly-OC2H5, where X is Leu, Ile, Val, Phe and Ala, have been prepared. Collagenolytic enzymes from various sources cleave these substrates with differing relative rate patterns. This series of peptides should be valuable for characterization of collagenases.     

A kinked antimicrobial peptide from <Emphasis Type="Italic">Bombina maxima</Emphasis>. II. Behavior in phospholipid bilayers

The preceding contribution by Toke et al. has studied the structure of the cationic antimicrobial peptide maximin-4 in detergent micelles and in organic solvent, revealing a different kink angle and side-chain interactions in the two different environments. Here, we have examined the same peptide in lipid bilayers using oriented circular dichroism (OCD) and solid-state ¹⁵N nuclear magnetic resonance (NMR) in aligned samples. OCD showed that maximin-4 is helical and adopts an oblique alignment in the membrane, and lacks the characteristic realignment response that is often observed for amphipathic α-helical peptides at a peptide:lipid ratio between 1:100 and 1:20. Solid-state ¹⁵N-NMR experiments suggest that maximin-4 also remains unaffected by lipid charge and temperature. Analyzing ¹⁵N labels in positions Ala12, Ala13, and Leu14, an oblique tilt angle of the N-terminal helix of ~130° relative to the membrane normal was found, in good agreement with the amphiphilic profile of this segment. An additional constraint at Ala22 in the C-terminal segment is found to be compatible with a continuous α-helix, but unfavorable side-chain interactions make this solution unlikely. Instead, a kink at Gly16 seems fully compatible with all known constraints and with the biophysical expectations in the membrane-bound state, given the liquid-state NMR structures. It thus seems that the flexible kink in maximin-4 allows the two helical segments to adjust to the local environment. The irregular amphiphilic profile and the resulting versatility in shape might explain why maximin-4 lacks the realignment response that has been characteristically observed for many related frog peptides forming straight amphipathic α-helices.     

Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments.

We prepared two dissected fragments of hen lysozyme and examined whether or not these two fragments associated to form a native-like structure. One (Fragment I) is the peptide fragment Asn59-homoserine-105 containing Cys64-Cys80 and Cys76-Cys94. The other (Fragment II) is the peptide fragment Lys1-homoserine-58 connected by two disulfide bridges, Cys6-Cys127 and Cys30-Cys115, to the peptide fragment Asn106-Leu129. It was found that the Fragment I immobilized in the cuvette formed an equimolar complex with Fragment II (K(d) = 3.3x10(-4) M at pH 8 and 25 degrees C) by means of surface plasmon resonance. Moreover, from analyses by circular dichroism spectroscopy and ion-exchange chromatography of the mixture of Fragments I and II at pH 8 under non-reducing conditions, it was suggested that these fragments associated to give the native-like structure. However, the mutant Fragment I in which Cys64-Cys80 and Cys76-Cys94 are lacking owing to the mutation of Cys to Ala, or the mutant fragment in which Trp62 is mutated to Gly, did not form the native-like species with Fragment II, because the mutant Fragment I derived from mutant lysozymes had no local conformation due to mutations. Considering our previous results where the preferential oxidation of two inside disulfide bonds, Cys64-Cys80 and Cys76-Cys94, occurred in the refolding of the fully reduced Fragment I, we suggest that the peptide region corresponding to Fragment I is an initiation site for hen lysozyme folding.     

Glycine and beta-branched residues support and modulate peptide helicity in membrane environments.

Transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation once inserted into the membrane, yet consist of primary sequences rich in residues known in soluble proteins as helix-breakers (Gly) and beta-sheet promoters (Ile, Val, Thr). To examine the specific 2 degrees structure propensities of such residues in membrane environments, we have designed and synthesized a series of 20-residue peptides with 'guest' hydrophobic segments--expected to provide three turns of incipient alpha-helix content--embedded in 'host' hydrophilic (Lys-Ser) matrices. Circular dichroism (CD) spectra of the model peptides in water showed that significant helical content was observed only for peptides with high Ala content; others behaved as 'random coils'. However, in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, it was found that Gly can be accommodated as readily as Ala, and Ile or Val as readily as Leu, in hydrophobic alpha-helices. Further subtleties of structural preferences could be observed in electrically-neutral lyso-phosphatidylcholine (LPC) micelles, where helical propensity decreased in the order Ala-Leu-rich > Gly-Leu-rich > Gly-Ile(Val)-rich hydrophobic segments. The results conjure a role of environment-dependent helix-modulation for Gly, Ile, and Val residues--and suggest that these residues may provide, in part, the structural basis for conformational transitions within or adjacent to membrane domains, such as those accompanying membrane insertion and/or required for transport or signalling functions.     

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees 相似文献   

The distal C-terminal region of the KcsA potassium channel is a pH-dependent tetramerization domain

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition. Here, we investigate the involvement of this domain in pH-mediated channel opening by NMR using a soluble peptide corresponding to residues 128-160 of the CTD (CTD34). At neutral pH, CTD34 exhibits concentration-dependent spectral changes consistent with oligomer formation. We prove this slowly tumbling species to be a tetramer with a dissociation constant of (2.0±0.5)×10(-)(11)?M(3) by NMR and sedimentation equilibrium experiments. Whereas monomeric CTD34 is only mildly helical, secondary chemical shifts prove that the tetrameric species adopts a tight native-like helical bundle conformation. The tetrameric species undergoes pH-dependent dissociation, and CTD34 is fully monomeric below pH?5.0. The structural basis for this phenomenon is the destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide. We conclude that (i) the CTD34 peptide is independently capable of forming a tetrameric helical bundle, and (ii) this structurally significant conformational shift is modulated by the effects of solution pH on residue H145. Therefore, the involvement of this domain in the pH gating of the channel is strongly suggested.