Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lai, J. R.
Right arrow Articles by Gellman, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lai, J. R.
Right arrow Articles by Gellman, S. H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
Protein Science (2003), 12:560-566.
Copyright © 2003 The Protein Society

Reinvestigation of the proposed folding and self-association of the Neuropeptide Head Activator

Jonathan R. Lai1 and Samuel H. Gellman1,2

1 Graduate Program in Biophysics, University of Wisconsin, Madison, Wisconsin 53706, USA
2 Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA

Reprint requests to: S.H. Gellman, Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706, USA; e-mail: gellman{at}chem.wisc.edu; fax: (608) 265-4534.

(RECEIVED September 9, 2003; FINAL REVISION November 21, 2002; ACCEPTED November 22, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0232103.


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 Electronic supplemental material
 References
 
The Neuropeptide Head Activator (HA), pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe (pGlu is pyroglutamic acid), is involved in head-specific growth and differentiation processes in the freshwater coelenterate Hydra attenuata. Peptides of identical sequence have also been isolated from higher-organism tissues such as human and bovine hypothalamus. Early studies by molecular sieve chromatography suggested that HA dimerizes with high affinity (Kd {approx} 1 nM). This dimerization was proposed to occur via antiparallel ß-sheet formation between the Lys7-Phe11 segments in each HA molecule. We conducted biophysical studies on synthetic HA in order to gain insight into its structure and aggregation tendencies. We found by analytical ultracentrifugation that HA is monomeric at low millimolar concentrations. Studies by 1H-NMR revealed that HA did not adopt any significant secondary structure in solution. We found no NOEs that would support the proposed dimer structure. We probed the propensity of the Lys7-Phe11 fragment to form antiparallel ß-sheet by designing peptides in which two such fragments are joined by a two-residue linker. These peptides were intended to form stable ß-hairpin structures with cross-strand interactions that mimic those of the proposed HA dimer interface. We found that the HA-derived fragments may be induced to form intramolecular ß-sheet, albeit only weakly, when linked by the highly ß-hairpin-promoting D-Pro-Gly turn, but not when linked by the more flexible Gly-Gly unit. These findings suggest that the postulated mode of HA dimerization and the proposed propensity of the molecule to form discrete aggregates with high affinity are incorrect.

Keywords: Neuropeptide Head Activator; ß-sheet; ß-hairpin; equilibrium analytical ultracentrifugation


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 Electronic supplemental material
 References
 
The Neuropeptide Head Activator (HA), pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe (pGlu is pyroglutamic acid), was first isolated in 1981 from the freshwater coelenterate Hydra attenuata (Schaller and Bodenmuller 1981), where it is involved in head-specific growth and differentiation processes (for review, see Schaller and Bodenmuller 1985; Schaller et al. 1989). Identical peptides have since been purified from higher-organism tissues such as human and bovine hypothalamus, and rat intestine (Bodenmuller and Schaller 1981). Early studies revealed that the purified peptide was active only at very low concentrations, a result that was later supported by analysis of chemically synthesized HA (Birr et al. 1981; Schaller and Bodenmuller 1981). Subsequent experiments by molecular sieve chromatography suggested that the peptide forms homodimers with high affinity (Kd {approx} 1 nM; Bodenmuller et al. 1986), which led to the hypothesis that inactivation at higher concentrations is due to self-association.

Previous attempts at conformational characterization of HA suggested that the molecule contains considerable ß-sheet structure (Bodenmuller et al. 1986; Saffrich et al. 1989; Fuentes et al. 1994). This hypothesis is unusual given that small linear peptides are generally too flexible to adopt well defined conformations in aqueous solution (Wright et al. 1988; Creighton 1996). Bodenmuller et al. (1986) proposed that homodimerization of the peptide occurs via an antiparallel sheet-like interaction. At the proposed dimer interface, residues Lys7 through Phe11 of one HA molecule are postulated to form an extended antiparallel ß-sheet with the same residues of a second HA molecule (Fig. 1AGo). The dimerization is supposedly driven by intermolecular hydrophobic interactions between sidechains on opposing strands and by a symmetrical pair of salt bridges between Lys7 of one HA molecule and the C-terminal carboxylate of the other.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. (A) Proposed HA homodimer interface (Bodenmuller et al. 1986), where Z is pyroglutamic acid. Postulated hydrogen bonds are indicated by dotted lines. The dimerization is proposed to be driven by specific contact between residues on opposing strands, and a symmetrical pair of salt-bridges between the {varepsilon}-amino of Lys7 on one HA molecule with the C-terminal carboxylate of the other HA molecule. (B) Hairpin conformations of peptides (HA-f)2-GG and (HA-f)2-pG that would mimic the interactions that are proposed to stabilize the dimer interface. Hydrogen bonding pattern between specific residues would remain in registry if residues 10 and 11 adopted a 2:2 reverse turn. In (HA-f)2-GG, X10 = Gly, and in (HA-f)2-pG, X10 = D-Pro.

 
This proposed interaction, if correct, would represent an important finding for the protein science community, because subnanomolar protein-protein interactions usually require extensive hydrophobic interactions involving burial of large surface areas between well folded protein components (Spolar and Record, Jr. 1994; Clackson and Wells 1995; Jones and Thornton 1996; Yao et al. 1998; DeLano 2002). In addition, there is growing interest in identifying peptides that can be used to bring other molecules (e.g., protein segments) together noncovalently (Arndt et al. 2000; Ghosh et al. 2000; Remy and Michnick 2001; Galarneau et al. 2002), and in development of small peptides or organic molecules that that can mimic the interfaces of protein-protein interactions (Peczuh and Hamilton 2000; Orner et al. 2001). In the present study, we reexamined the HA dimerization hypothesis via several experimental strategies.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 Electronic supplemental material
 References
 
Peptide design
Several peptides were designed and synthesized to examine the aggregational and conformational tendencies of HA-related sequences. The natural HA peptide (with the sequence ZPPGGSKVILF-CO2-, where the residues involved in the putative dimer interface are underlined) was synthesized and includes the modified glutamate residue (pyroglutamic acid, Z) at its N-terminus. The peptide HA-f (fragment) consists of the C-terminal six residues of HA (sequence +NH3-SKVILF-CO2-). This fragment has been reported to dimerize with even higher affinity than does the full-length peptide (Bodenmuller et al. 1986). In the proposed dimer interface, five of the six residues of HA-f (underlined) are involved in the antiparallel ß-sheet formed between the two HA molecules. Therefore, HA-f represents the minimal sequence required for the proposed self-association.

Figure 1AGo shows the postulated mode of dimerization in which residues Lys7 though Phe11 of one HA molecule form an antiparallel two-stranded ß-sheet with the same residues of another HA molecule. We hypothesized that these interactions could be mimicked in an intramolecular fashion (Fig. 1BGo), and the validity of the proposed dimer interface could be tested by probing the propensity of the resulting peptide to form a ß-hairpin. This configuration could be achieved by linking two segments corresponding to the C-terminal five residues of HA with a dipeptide loop. The propensity of the resulting peptides to form ß-hairpins is easily evaluated using biophysical techniques and provides a strategy to detect interactions without complications due to nonspecific aggregation. Peptides (HA-f)2-GG and (HA-f)2-pG were designed in this fashion. Fragments of HA were linked with either a flexible Gly-Gly loop [(HA-f)2-GG has the sequence +NH3-RRGSKVILF-GG-KVILF-CO2-], or a ß-hairpin-promoting D-Pro-Gly loop [(HA-f)2-pG has the sequence +NH3-RRGSKVILF-pG-KVILF-CO2-, where the lowercase letter denotes D absolute configuration]. An Arg-Arg-Gly-Ser segment was included at the N-termini of these peptides to improve solubility and discourage aggregation. The C-termini were left free to allow a potential electrostatic interaction between the carboxy group and the Lys5 sidechain on the opposing strand. A similar interaction is postulated to be important for HA dimerization (Bodenmuller et al. 1986).

A ß-hairpin with specific residue interactions mimicking those of the proposed interface could be envisioned if the Gly-Gly linker region and flanking residues of (HA-f)2-GG adopted a 2:2 ß-hairpin loop (see Fig. 1Go; Sibanda et al. 1989). ß-Hairpin formation with this type of loop, in which the two loop residues correspond to residues i+1 and i+2 of a ß-turn, would maintain the registry of the hydrogen bond network of the postulated intermolecular HA dimer interface and would allow the same specific interactions that are proposed to stabilize the HA dimer. Peptide (HA-f)2-pG is very similar to (HA-f)2-GG, but the flexible Gly-Gly linker has been replaced by the highly hairpin-promoting D-Pro-Gly linker. The D-Pro-Gly segment strongly favors formation of type I‘ and type II‘ ß-turns (as residues i+1 and i+2). These unusual types of ß-turn are commonly observed at the loops of 2:2 ß-hairpins in proteins (Wilmot and Thornton 1988). The D-Pro-Gly segment has been shown to promote ß-hairpin formation in several short designed peptides (Haque et al. 1996; Haque and Gellman 1997; Stanger and Gellman 1998; Das et al. 2001). Furthermore, the incorporation of D-Pro into reverse turns has also been employed in several instances to stabilize antiparallel ß-sheet interactions between short strand segments derived from natural proteins outside the context of the natural tertiary structure (Struthers et al. 1996; Haque and Gellman 1997; Espinosa and Gellman 2000; Kaul et al. 2001). The Gly-Gly segment in (HA-f)2-GG endows the linker with high flexibility, and any ß-hairpin structure observed for (HA-f)2-GG would be attributable largely to the propensity of the strand segments to form cross-strand contacts that mimic those of the postulated HA dimer interface. In contrast, the D-Pro-Gly segment of (HA-f)2-pG strongly promotes formation of the ß-hairpin by enforcing an appropriate reverse turn. In this case, even modest interstrand contacts should be sufficient to induce folding. The ß-hairpin configurations of the designed peptides that would mimic the proposed HA dimer interface are presented in Figure 1BGo.

Aggregational and conformational tendencies of HA and HA-f
Equilibrium analytical ultracentrifugation (AU) at concentrations of 1.0 mM and 1.3 mM in aqueous 50 mM acetic acid, pH 5.0, revealed that HA was predominantly monomeric under these conditions (Fig. 2Go). The observation that HA is monomeric at low millimolar concentrations contradicts earlier conclusions based on molecular sieve chromatography, in which HA was thought to elute predominantly as a dimer at concentrations as low as 10-11 M (Bodenmuller et al. 1986). In these initial studies, the dimer was thought to be stable under aqueous buffering conditions such as 0.01 M to 0.1 M Tris (pH 7.5) containing 0.1 M NaCl [the authors reported that extremely harsh conditions such as 1 M (NH4)SO4 or boiling the peptide in 0.1 M HCl were required to obtain exclusively monomeric HA]. Although our analysis of HA was performed at pH 5.0, we conclude that there are no differences in conformation or aggregation state between our sample and that previously analyzed by NMR at pH 7.2 by Saffrich et al. (1989) because the chemical shifts are identical (discussed below). From our AU studies, we observe no traces of a discrete HA dimer with high association affinity.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. Representative equilibrium analytical ultracentrifugation for 1.0 mM HA in aqueous 50 mM acetic acid, pH 5.0 at 297 K. Absorbance data were acquired at 257 nm. Presence of a single species is indicated by linearity of the data. The slope is directly proportional to the molecular weight. Linear least-square regression analysis yielded molecular weight estimates consistent with HA monomer. The data shown were acquired at a rotor speed of 56 krpm, resulting in an experimental mass of ~1024 g mole-1 (theoretical mass of HA is 1126 g mole-1). Discrepancies between observed and predicted masses may be due to nonideality effects. The fits were judged as adequate by randomness of the residuals about zero (top). Fit is shown as a solid line. Theoretical plots for monomer and dimer are also shown, as dashed lines.

 
The peptide HA-f was reported to form a dimer with even higher affinity than HA (Bodenmuller et al. 1986). However, our analysis by AU revealed that HA-f forms large, nondiscrete aggregates at several concentrations ranging from 0.9 mM to 2.0 mM (see Electronic Supplemental Material). This behavior is not surprising based on the high content of hydrophobic and ß-branched residues in HA-f. ß-Branched residues have high propensity for extended ß-strand conformation (Chou and Fasman 1974 Chou and Fasman 1978; Kim and Berg 1993; Minor and Kim 1994), and the hydrophobic sidechains of the Val8, Ile9, Leu10, and Phe11 residues provide a driving force for self-association. Because hydrogen bond networks could be formed on either side of HA-f if the molecule were to adopt an extended conformation, the sequence may be prone to formation of infinitely large ß-sheet aggregates (for examples of ß-sheet self-assembly, see Lashuel et al. 2000; Powers and Kelly 2001; Powers et al. 2002). However, it is doubtful that this type of aggregation would involve specific residue-to-residue interactions as postulated for the HA dimer interface. Nonspecific aggregation is known to occur with peptides containing a similar sequence, Lys-Leu-Val-Phe-Phe, which is believed to be involved in amyloid fibril formation (Burkoth et al. 2000; Ramirez-Alvarado et al. 2000; Cairo et al. 2002; Kelly 2002).

The 1H-NMR spectra for HA were well resolved, and full TOCSY (Bax and Davis 1985) spin-system assignments were achieved using NOESY (Jeener et al. 1979) and ROESY (Bothner-By et al. 1984) experiments (complete chemical shift assignments are found in the Supplemental Material). The observed 1H chemical shifts for HA were in excellent agreement with previous NMR analysis of HA (Saffrich et al. 1989). The maximum chemical shift differences were 0.01 ppm, that is, within experimental uncertainty, for all amide protons and {alpha}-protons [these types of protons are highly sensitive to secondary structure (Wishart et al. 1991Wishart et al. 1992)]. The similarities in chemical shifts observed for HA with previously reported values for HA suggest that there are no conformational differences between our sample and that analyzed previously in unbuffered aqueous solutions (Saffrich et al. 1989).

Chemical shifts for {alpha}-protons of residues in a ß-sheet conformation are generally shifted downfield relative to {alpha}-protons of residues in the random coil state, while {alpha}H chemical shifts of residues in {alpha}-helical conformations are shifted upfield relative to random coil (Wishart et al. 1991Wishart et al. 1992). Three consecutive residues with consistent deviations from random coil larger than 0.1 ppm are considered evidence of secondary structure formation (Wishart et al. 1992). Figure 3AGo shows the deviation of observed {alpha}-proton chemical shifts from published random coil values [{delta}{alpha}H(observed) - {delta}{alpha}H(random coil); random coil values from Wuthrich 1986] for HA. These data suggest that HA is predominantly in a random coil state under these conditions, a finding that contradicts secondary structural predictions that HA forms 62%–67% ß-sheet conformation (Bodenmuller et al. 1986; Fuentes et al. 1994).




View larger version (42K):
[in this window]
[in a new window]
  		Figure 3. (A) Deviation of {alpha}-proton chemical shifts for HA from random coil values (Wuthrich 1986) plotted against residue. Literature random coil values for pGlu are not available, therefore this residue is not plotted. The datum for the Phe11 is also not plotted due to effects from the C-terminal carboxylate. The lack of consistently large deviations from random coil ({delta}{delta}{alpha}H > 0.1 ppm) implies that the peptide does not adopt significant secondary structure. Analysis was performed at 277 K in 90% H2O/10% D2O containing 100 mM acetic acid-d6, pH 5.0 (uncorrected). (B) Chemical shift analysis for regions of (HA-f)2-GG (open bars) and (HA-f)2-pG (solid bars) that contain the HA-related sequences. Data acquired under the conditions described in A. Turn positions [Xaa10 and Gly11, where Xaa = Gly in (HA-f)2-GG and Xaa = D-Pro in (HA-f)2-pG] are not plotted. Data for Phe16 of both peptides are also not included due to C-terminal carboxylate effects. Significant positive {alpha}-proton chemical shift deviations from random coil for (HA-f)2-pG suggest some ß-sheet content.

 
Several NOEs were observed for HA, but none is consistent with the proposed ß-sheet-like interface. Antiparallel ß-sheets show several characteristic NOE patterns including crosspeaks between {alpha}-protons of residues in nonhydrogen-bonded positions on opposite strands (e.g., Val8 {alpha}H to Leu10 {alpha}H in Fig. 1AGo), and crosspeaks between amide protons of residues hydrogen bonded to one another on opposite strands (e.g., Ile5 NH to Ile9 NH in Fig. 1AGo). None of these characteristic NOEs was observed for HA. Of the interresidue NOEs that could be unambiguously assigned, only two were between sequentially nonadjacent residues. Both of these signals were of weak intensity and between protons on Ile9 and Phe11. Two other signals may have been between sequentially nonadjacent residues but could not be unambiguously assigned due to spectral overlap (one between an Ile9 sidechain proton and either Ser6 NH or Leu10 NH, and another between Phe11 NH and either Ser6 NH or Leu10 NH). From this analysis it is not possible to distinguish inter- from intramolecular NOEs. Because we find by AU that HA is monomeric under similar conditions (NMR analysis performed at ~1 mM), it is unlikely that these NOEs are due to self-association. If these NOEs were due to intramolecular contacts, these data would suggest partial folding of the peptide to a conformation with specific interactions. However, the paucity and low intensity of the nonsequential NOEs suggests that the conformation giving rise to these NOEs is very sparsely populated. These NOE data suggest that the postulated head-to-tail mode of HA dimerization is incorrect.

Evaluation of the proposed HA dimer interface via structural analysis of (HA-f)2-GG and (HA-f)2-pG
Our 1H-NMR analysis provides no indication of significant ß-hairpin formation by (HA-f)2-GG in solution. As shown in Figure 3BGo, no systematic deviation of {alpha}-proton chemical shifts from random coil values was observed for any region of the peptide. Furthermore, no nonsequential NOEs were observed for (HA-f)2-GG. Geminal {alpha}-proton splitting for glycine residues in ß-turns has been used as an indication of ß-hairpin formation (Searle et al. 1999; Griffith-Jones and Searle 2000). For (HA-f)2-GG, the Gly9 protons were split very slightly (36 Hz), and chemical shifts for geminal protons of Gly10 were only moderately different (by 96 Hz). Vicinal 3J{alpha}N proton couplings are also frequently used as indicators of secondary structure for proteins and peptides (Serrano 1995; Smith et al. 1996; Griffiths-Jones et al. 1998). Large values (> 8.0 Hz) are indicative of ß-sheet structure, whereas smaller values (< 6.0 Hz) signify helical conformation. Most of the 3J{alpha}N could not be determined from the proton 1D-spectrum of (HA-f)2-GG due to poor dispersion; however, of the four values that could be extracted, none was above 8.0 Hz (Ser4, 6.8 Hz; Leu8, 7.7 Hz; Lys12, 7.0 Hz; Phe16, 7.9 Hz). Taken together, these data indicate that (HA-f)2-GG does not adopt the ß-hairpin conformation depicted in Figure 1BGo.

NMR evidence suggests that peptide (HA-f)2-pG is at least partially folded into a ß-hairpin, in contrast to (HA-f)2-GG. Several of the {alpha}-proton chemical shifts for residues near the turn (Val6–Phe9 and Leu12–Val13) are suggestive of ß-sheet conformation (Fig. 3BGo). In addition, several NOEs consistent with ß-hairpin formation were observed (Fig. 4Go), including a long-range interaction between the {alpha}-protons of Leu8 and Val13. An additional {alpha}-proton-to-{alpha}-proton NOE was detected, but this signal could not be assigned unambiguously due to spectral overlap (Leu15 {alpha}H to either Val6 {alpha}H or Ile7 {alpha}H). Two turn-defining NH-to-NH NOEs were also observed, one between the amide protons of Phe9 and Lys12, and another between Gly11 and Lys12. The amide 1H signals were well dispersed, and the majority of the 3J{alpha}N that could be resolved were indicative of significant ß-sheet population (Lys5, 7.2 Hz; Ile7, 9.2 Hz; Leu8, 9.1 Hz; Phe9, 7.0 Hz; Lys12, 8.1 Hz; Val13, 7.1 Hz; Ile14, 9.2 Hz; Phe16, 8.0 Hz). The turn glycine geminal {alpha}-proton signals were split by 150 Hz. These data suggest that (HA-f)2-pG is at least partially folded in the intended ß-hairpin conformation, particularly in the turn region. This observation implies that the linked HA fragments may be induced to associate in a ß-sheet mode if they are connected by a linker with a high turn propensity. However, this effect results in a ß-hairpin that is only moderately stable, indicating that the cross-strand interactions are relatively weak. Based on the behavior of (HA-f)2-GG and (HA-f)2-pG, we conclude that the potential for interaction between the two HA-derived fragments exists, but that this interaction would not be adequate to drive dimerization at low concentration. Furthermore, we conclude that the postulated mode of HA dimerization and the strength of the aggregation as earlier reported are incorrect.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Long-range NOEs from 200 msec NOESY and ROESY spectra of (HA-f)2-pG that are consistent with ß-hairpin formation. One signal could not be unambiguously assigned due to spectral overlap (between the {alpha}-protons of Leu15 and Val6 or Ile7) and is indicated by a double-headed arrow. Extremely weak NOEs are depicted by dotted lines.

 

   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
Electronic supplemental material
 References
 
Peptide synthesis and purification
All peptides were synthesized on an Applied Biosystems Model 432A Synergy automated synthesizer using standard solid-phase Fmoc chemistry with HBTU activation. The sidechain protecting groups were butoxycarbonyl (Boc) for lysine, t-butyl for serine and threonine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) for arginine, and trityl for glutamine (all amino acids were from NovaBiochem, except for L-pyroglutamic acid, from Aldrich). The peptides were synthesized as C-terminal free acids on a methylbenzlhydrylamine (MBHA) polystyrene resin functionalized with a 4-hydroxy-methylphenoxy (HMP) linker (Applied Biosystems). Following synthesis, simultaneous cleavage from support and sidechain deprotection was achieved by treatment with a cocktail containing 85% (v/v) trifluoroacetic acid (TFA), 5% (v/v) 1,2-ethanedithiol, and 10% (v/v) thioanisole. Peptides were purified by reverse-phase HPLC (Vydac silica-C4 or -C18 semipreparative columns, 5 µm, 250 mm x 10 mm) with water/0.1% TFA and water/acetonitrile/0.1% TFA solution mobile phases. Homogeneity of final samples was verified by analytical HPLC (Vydac silica-C4 column, 5 µm, 250 mm x 4 mm). Identity of isolated compounds was confirmed using matrix-assisted laser-desorption/ionization time-of-flight spectrometry (MALDI-TOF, measurements performed on a Bruker REFLEX II instrument).

Equilibrium analytical ultracentrifugation (AU)
Samples were prepared by dissolving the pure lyophilized peptides, isolated as the TFA salt, to the appropriate concentration in aqueous 50 mM acetic acid, pH 5.0. Equilibrium ultracentrifugation studies were conducted on a Beckman XLA ultracentrifuge at 297 K. Cells with 1.2 cm pathlengths were used, and the absorbance was monitored at 257 nm. Analysis was performed at several rotor speeds ranging from 40 to 60 krpm. Data were acquired with a 0.001 cm step size at each speed every 4 h until three consecutive spectra were identical. Linear least-squares fitting was performed in accordance with the equation d (ln c)/d r2 = M(1-{nu}{rho}){omega}2/2RT, where d (ln c)/d r2 is the slope of a ln(Absorbance) versus (Radial Distance)2 plot, {rho} is the partial specific volume, {rho} is the solvent density, {omega} is the rotor speed, R is the gas constant, and T is temperature (Cantor and Schimmel 1980; Schuster and Laue 1994). Molecular weight estimates were determined from the parameter M. A partial specific volume of 0.786 mL g-1 for HA was calculated by the method of Durchschlag and Zipper (1994). A solvent density of 0.99885 g mL-1 was determined at 297 K using an Anton Parr DMA 5000 density meter.

Nuclear magnetic resonance (NMR)
NMR samples were prepared by dissolving the lyophilized peptides to ~1 to 2 mM in 10% D2O/90% H2O or pure D2O, containing 100mM acetic acid-d6, pH 5.0 (uncorrected). Spectra were acquired on a Varian INOVA 600 MHz spectrometer at probe temperatures ranging from 277 K to 303 K as necessary to resolve resonance overlap. A 7000 Hz spectral window was used for all acquisitions, with 80 msec, 200 msec, and 200 msec mix times for TOCSY (Bax and Davis 1985), ROESY (Bothner-By et al. 1984), and NOESY (Jeener et al. 1979) experiments, respectively. Solvent suppression in the TOCSY and ROESY spectra was achieved using a selective presaturation pulse, and in NOESY with the WET method (Ogg et al. 1994; Smallcombe et al. 1995). Typical data sets consisted of 500 to 600 free-induction decay increments of 16 to 24 transients each (corresponding to 2048 to 4096 points in f2). All spectra were processed in standard Varian software, and referenced to internal 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) standard. Although analytical ultracentrifugation was not performed on (HA-f)2-GG or (HA-f)2-pG, we assume that NMR studies on these peptides were conducted under nonaggregating conditions, because a 10-fold dilution of the NMR samples yielded identical one-dimensional spectra. It is unlikely that these peptides would aggregate below this concentration range (0.1 to 0.2 mM) due to their high charge density (overall charge at pH 5.0 should be +4).


   Electronic supplemental material
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 Electronic supplemental material
References
 
Additional ultracentrifugation data for HA and HA-f may be found in the Supplemental Materials. Full chemical shift assignments, and a complete list of NOEs from the 1H-NMR analysis of HA, are also included.


   Acknowledgments
 
This work was supported by the NIH (GM 61238). J.R.L. is supported in part by a PGS B scholarship from the Natural Sciences and Engineering Research Council (NSERC) of Canada.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 Electronic supplemental material
 References
 
Arndt, K.M., Pelletier, J.N., Muller, K.M., Alber, T., Michnick, S.W., and Pluckthun, A. 2000. A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble. J. Mol. Biol. 295: 627–639.[CrossRef][Medline]

Bax, A. and Davies, D.G. 1985. MLEV-17 based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65: 355–360.

Birr, C., Zachmann, B., Bodenmuller, H., and Schaller, H.C. 1981. Synthesis of a new neuropeptide, the head activator from hydra. FEBS Lett. 131: 317–321.[CrossRef][Medline]

Bodenmuller, H. and Schaller, H.C. 1981. Conserved amino acid sequence of a neuropeptide, the head activator, from coelenterates to humans. Nature 293: 579–580.[CrossRef][Medline]

Bodenmuller, H., Schilling, E., Zachmann, B., and Schaller, H.C. 1986. The neuropeptide head activator loses its biological activity by dimerization. EMBO J. 5: 1825–1829.[Medline]

Bothner-By, A.A., Stephens, R.L., Lee, J., Warren, C.D., and Jeanloz, R.W. 1984. Structure determination of a tetrasaccharide: Transient nuclear overhauser effects in the rotating frame. J. Am. Chem. Soc. 106: 811–813.[CrossRef]

Burkoth, T.S., Benzinger, T.L.S., Urban, V., Morgan, D.M., Gregory, D.M., Thiyagarajan, P., Botto, R.E., Meredith, S.C., and Lynn, D.G. 2000. Structure of the ß-amyloid(10–35) fibril. J. Am. Chem. Soc. 122: 7883–7889.[CrossRef]

Cairo, C.W., Strzelec, A., Murphy, R.M., and Kiessling, L.L. 2002. Affinity-based inhibition of ß-amyloid toxicity. Biochemistry 41: 8620–8629.[CrossRef][Medline]

Cantor, C.R. and Schimmel, P.R. 1980. Biophysical chemistry: Techniques for the study of biological structure and function, Vol. II, pp. 590–641. W.H. Freeman and Company, New York.

Chou, P.Y. and Fasman, G.D. 1974. Conformational parameters for amino acids in helical, ß-sheet, and random coil regions calculated from proteins. Biochemistry 13: 211–222.[CrossRef][Medline]

———. 1978. Empirical predictions of protein conformation. Annu. Rev. Biochem. 47: 251–276.[CrossRef][Medline]

Clackson, T. and Wells, J.A. 1995. A hot spot of binding energy in a hormone-receptor interface. Science 267: 383–386.[Abstract/Free Full Text]

Creighton, T.E. 1996. Proteins: Structures and molecular properties, 2nd ed., pp. 139–259. W.H. Freeman and Company, New York.

Das, C., Naganagowda, G.A., Karle, I.L., and Balaram, P. 2001. Designed ß-hairpin peptides with defined tight turn stereochemistry. Biopolymers 58: 335–346.[CrossRef][Medline]

DeLano, W.L. 2002. Unraveling hot spots in binding interfaces: Progress and challenges. Curr. Opin. Struct. Biol. 12: 14–20.[CrossRef][Medline]

Durchschlag, H., and Zipper, P. 1994. Calculation of the partial specific volume of organic compounds and polymers. Prog. Colloid Polym. Sci. 95: 20–39.

Espinosa, J.F. and Gellman, S.H. 2000. A designed ß-hairpin containing a natural hydrophobic cluster. Angew. Chem. Int. Ed. Engl. 39: 2330–2333.[CrossRef][Medline]

Fuentes, E.J., Pachter, R., and Tsonis, P.A. 1994. On the three-dimensional structure of the hydra head activator neuropeptide. In Vivo 8: 199–206.[Medline]

Galarneau, A., Primeau, M., Trudeau, L.-E., and Michnick, S.W. 2002. ß-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein–protein interactions. Nat. Biotechnol. 20: 619–622.[CrossRef][Medline]

Ghosh, I., Hamilton, A.D., and Regan, L. 2000. Antiparallel leucine zipper-directed protein reassembly: Application to the green fluorescent protein. J. Am. Chem. Soc. 122: 5658–5659.[CrossRef]

Griffiths-Jones, S.R. and Searle, M.S. 2000. Structure, folding, and energetics of cooperative interactions between the ß-strands of a de novo designed three-stranded antiparallel ß-sheet peptide. J. Am. Chem. Soc. 122: 8350–8356.[CrossRef]

Griffiths-Jones, S.R., Sharman, G.J., Maynard, A.J., and Searle, M.S. 1998. Modulation of intrinsic {phi}, {xi} propensities of amino acids by neighboring residues in the coil regions of protein structures: NMR analysis and dissection of a ß-hairpin peptide. J. Mol. Biol. 284: 1597–1609.[CrossRef][Medline]

Haque, T.S. and Gellman, S.H. 1997. Insights on ß-hairpin stability in aqueous solution from peptides with enforced type I‘ and type II‘ ß-turns. J. Am. Chem. Soc. 119: 2303–2304.[CrossRef]

Haque, T.S., Little, J.S., and Gellman, S.H. 1996. Stereochemical requirements for ß-hairpin formation: Model studies with four-residue peptides and depsipeptides. J. Am. Chem. Soc. 118: 6975–6985.[CrossRef]

Jeener, J., Meier, B.H., Bachmann, R., and Ernst, R.R. 1979. Investigation of exchange processes by two-dimensional NMR spectroscopy. J. Chem. Phys. 71: 4546–4553.[CrossRef]

Jones, S. and Thornton, J.M. 1996. Principles of protein–protein interactions. Proc. Natl. Acad. Sci. 93: 13–20.[Abstract/Free Full Text]

Kaul, R., Angeles, A.R., Jager, M., Powers, E.T., and Kelly, J.W. 2001. Incorporating ß-turns and a turn mimetic out of context in loop 1 of the WW domain affords cooperatively folded ß-sheets. J. Am. Chem. Soc. 123: 5206–5212.[CrossRef][Medline]

Kelly, J.W. 2002. Towards an understanding of amyloidogenesis. Nat. Struct. Biol. 9: 323–325.[CrossRef][Medline]

Kim, C.A. and Berg, J.M. 1993. Thermodynamic ß-sheet propensities measured using a zinc-finger host peptide. Nature 362: 267–270.[CrossRef][Medline]

Lashuel, H.A., LaBrens, S.R., Woo, L., Serpell, L.C., and Kelly, J.W. 2000. Protofilaments, filaments, ribbons, and fibrils from peptidomimetic self-assembly: Implications for amyloid fibril formation and materials science. J. Am. Chem. Soc. 122: 5262–5277.[CrossRef]

Minor, D.L. and Kim, P.S. 1994. Measurement of the ß-sheet-forming propensities of amino acids. Nature 367: 660–663.[CrossRef][Medline]

Ogg, R.J., Kingsley, P.B., and Taylor, J.S. 1994. WET, a T1- and B1-insensitive water-suppression method for in vivo localized 1H-NMR spectroscopy. J. Magn. Reson. B. 104: 1–10.[CrossRef][Medline]

Orner, B.P., Ernst, J.T., and Hamilton, A.D. 2001. Toward proteomimetics: Terphenyl derivatives as structural and functional mimics of extended regions of an {alpha}-helix. J. Am. Chem. Soc. 123: 5382–5383.[CrossRef][Medline]

Peczuh, M.W. and Hamilton, A.D. 2000. Peptide and protein recognition by designed molecules. Chem. Rev. 100: 2479–2494.[CrossRef][Medline]

Powers, E.T. and Kelly, J.W. 2001. Medium-dependent self-assembly of an amphiphilic peptide: Direct observation of peptide phase domains at the air–water interface. J. Am. Chem. Soc. 123: 775–776.[CrossRef][Medline]

Powers, E.T., Yang, S.I., Lieber, C.M., and Kelly, J.W. 2002. Ordered Langmuir-Blodgett films of amphiphilic ß-hairpin peptides imaged by atomic force spectroscopy. Angew. Chem. Int. Ed. Engl. 41: 127–130.[CrossRef][Medline]

Ramirez-Alvarado, M., Merkel, J.S., and Regan, L. 2000. A systematic exploration of the influence of the protein stability on amyloid fibril formation in vitro. Proc. Natl. Acad. Sci. 97: 8979–8984.[Abstract/Free Full Text]

Remy, I. and Michnick, S.W. 2001. Visualization of biochemical networks in living cells. Proc. Natl. Acad. Sci. 98: 7678–7683.[Abstract/Free Full Text]

Saffrich, R., Kalbitzer, H.R., Bodenmuller, H., Muhn, P., Pipkorn, R., and Schaller, H.C. 1989. 1H-Nuclear magnetic resonance studies of the neuropeptide head activator. Biochim. Biophys. Acta. 997: 144–153.[CrossRef][Medline]

Schaller, H.C. and Bodenmuller, H. 1981. Isolation and amino acid sequence of a morphogenic peptide from hydra. Proc. Natl. Acad. Sci. 78: 7000–7004.[Abstract/Free Full Text]

———. 1985. Role of the neuropeptide head activator for nerve function and development. Biol. Chem. 366: 1003–1007.

Schaller, H.C., Hoffmeister, S.A.H., and Dubel, S. 1989. Role of the neuropeptide head activator for growth and development in hydra and mammals. Development (Suppl.): 99–107.

Schuster, T.M. and Laue, T.M., eds. 1994. Modern analytical ultracentrifugation: Acquisition and interpretation of data for biological and synthetic polymer systems. Birkhauser, Boston.

Searle, M.S., Griffiths-Jones, S.R., and Skinner-Smith, H. 1999. Energetics of weak interactions in a ß-hairpin peptide: Electrostatic and hydrophobic contributions to stability from lysine salt bridges. J. Am. Chem. Soc. 121: 11615–11620.[CrossRef]

Serrano, L. 1995. Comparison between the {phi} distribution of the amino acids in the protein database and NMR data indicates that amino acids have various {phi} propensities in the random coil conformation. J. Mol. Biol. 254: 322–333.[CrossRef][Medline]

Sibanda, B.L., Blundell, T.L., and Thornton, J.M. 1989. Conformation of ß-hairpins in protein structures: A systematic classification with applications to modeling by homology, electron density, and protein engineering. J. Mol. Biol. 206: 759–777.[CrossRef][Medline]

Smallcombe, S.H., Patt, S.L., and Keifer, P.A. 1995. WET solvent suppression and its applications to LC NMR and high-resolution NMR spectroscopy. J. Magn. Reson. A. 117: 295–303.[CrossRef]

Smith, L.J., Bolin, K.A., Schwalbe, H., MacArthur, M.W., Thornton, J.M., and Dobson, C.M. 1996. Analysis of main-chain torsion angles in proteins—Prediction of NMR coupling constants for native and random coil configurations. J. Mol. Biol. 255: 494–506.[CrossRef][Medline]

Spolar, R.S. and Record, Jr., M.T. 1994. Coupling of local folding to site-specific binding of proteins to DNA. Science 263: 777–784.[Abstract/Free Full Text]

Stanger, H.E. and Gellman, S.H. 1998. Rules for antiparallel ß-sheet design: D-Pro-Gly is superior to L-Asn-Gly for ß-hairpin nucleation. J. Am. Chem. Soc. 120: 4236–4237.[CrossRef]

Struthers, M.D., Cheng, R.P., and Imperiali, B. 1996. Design of a monomeric 23-residue polypeptide with defined tertiary structure. Science 271: 342–345.[Abstract]

Wilmot, C.M. and Thornton, J.M. 1988. Analysis and prediction of different types of ß-turn in proteins. J. Mol. Biol. 203: 221–232.[CrossRef][Medline]

Wishart, D.S., Sykes, B.D., and Richards, F.M. 1991. Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J. Mol. Biol. 222: 311–333.[CrossRef][Medline]

———. 1992. The chemical shift index: A fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 31: 1647–1651.[CrossRef][Medline]

Wright, P.E., Dyson, H.J., and Lerner, R.A. 1988. Conformation of peptide fragments of proteins in aqueous solution: Implications for initiation of protein folding. Biochemistry 27: 7167–7175.[CrossRef][Medline]

Wuthrich, K. 1986. NMR of proteins and nucleic acids. Wiley, New York.

Yao, S., Brickner, M., Pares-Matos, E.I., and Chmielewski, J. 1998. Uncoiling c-Jun coiled coils: Inhibitory effects of truncated Fos dimerization and DNA binding in vitro. Biopolymers 47: 277–283.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lai, J. R.
Right arrow Articles by Gellman, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lai, J. R.
Right arrow Articles by Gellman, S. H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS