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Effect of the N1 residue on the stability of the -helix for all 20 amino acids

Department of Biomolecular Sciences, UMIST, Manchester M60 1QD, UK

Reprint requests to: Dr. Andrew J. Doig, Department of Biomolecular Sciences, UMIST, Manchester M60 1QD, UK; e-mail: Andrew.Doig{at}umist.ac.uk; fax: 44-161-236-0409.

(RECEIVED July 27, 2000; FINAL REVISION November 20, 2000; ACCEPTED November 28, 2000)

Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.31001.

	Abstract

TOP Abstract Introduction Results and discussion Conclusion Materials and methods References

 
N1 is the first residue in an -helix. We have measured the contribution of all 20 amino acids to the stability of a small helical peptide CH₃CO-XAAAAQAAAAQAAGY-NH₂ at the N1 position. By substituting every residue into the N1 position, we were able to investigate the stabilizing role of each amino acid in an isolated context. The helix content of each of the 20 peptides was measured by circular dichroism (CD) spectroscopy. The data were analyzed by our modified Lifson-Roig helix-coil theory, which includes the n1 parameter, to find free energies for placing a residue into the N1 position. The rank order for free energies is Asp^-, Ala > Glu^- > Glu⁰ > Trp, Leu, Ser > Asp⁰, Thr, Gln, Met, Ile > Val, Pro > Lys⁺, Arg, His⁰ > Cys, Gly > Phe > Asn, Tyr, His⁺. N1 preferences are clearly distinct from preferences for the preceding N-cap and -helix interior. pK_a values were measured for Asp, Glu, and His, and protonation-free energies were calculated for Asp and Glu. The dissociation of the Asp proton is less favorable than that of Glu, and this reflects its involvement in a stronger stabilizing interaction at the N terminus. Proline is not energetically favored at the -helix N terminus despite having a high propensity for this position in crystal structures. The data presented are of value both in rationalizing mutations at N1 -helix sites in proteins and in predicting the helix contents of peptides.

Keywords: -Helix; N1 position; circular dichroism; protein folding; protein stability; helix propensities; helix-coil theory; macrodipole

	Introduction

TOP Abstract Introduction Results and discussion Conclusion Materials and methods References

 
The -helix is the most commonly occurring secondary structural element in proteins. Positional preferences for amino acids in the interior positions of the -helix have been measured experimentally in peptides and proteins (for review, see Pace and Scholtz 1998). Amino acid preferences for the helix differ greatly between terminal and interior positions (Argos and Palau 1982; Richardson and Richardson 1988; Kumar and Bansal 1998; Penel et al. 1999). The N terminus of an -helix has three amide hydrogens that lack the i,i+4 backbone hydrogen bonds that characterize the -helix; similarly, the C terminus has three free carbonyl groups. Presta and Rose (1988) suggested that hydrogen bonds from polar side chains to these otherwise unsatisfied hydrogen bonding partners are of critical importance in establishing the locations of -helix termini. The residue immediately preceding the N-terminal side of the -helix has been dubbed the N-cap, while the residue immediately following the C-terminal side of the -helix is the C-cap. The capping residues have nonhelical , dihedral angles, although they form helical i,i +4 hydrogen bonds. Between the capping residues lie the residues with -helical , dihedral angles, designated as N1, N2, N3 . . . C3, C2, and C1. While the N terminus of the -helix is structurally well characterized (Penel et al. 1999), it is less well understood in thermodynamic terms, and this represents one barrier to successfully predicting secondary structure from primary sequence. The N-cap, N1, N2, and N3 residues can potentially interact with the -helix macrodipole, free backbone amides, and local side chains in different ways and, thus, contribute differently to -helix stability.

While the Serrano group and our group have measured the free energy changes that arise when certain residues are substituted at N1 positions (Petukhov et al. 1998, 1999; Sun et al. 2000), many amino acids have not been investigated, notably the charged residues. In this article, we determine the intrinsic preferences for N1 positions in our -helical model peptide for all 20 naturally occurring amino acids. We use the methodology of previous work, where the N1 preferences of 11 amino acids were determined using new helix-coil theory (Sun et al. 2000) and previously published peptide data (Chakrabartty et al. 1993a). Here, we study 20 peptides of improved design that differ only in their N1 amino acid.

	Results and discussion

TOP Abstract Introduction Results and discussion Conclusion Materials and methods References

 
Helix-coil theory
-Helices in aqueous solution adopt a large number of structures, with fully helical, fully coil, and partly helical conformations all populated. For a complete understanding of helix formation and stability, all the factors contributing to this equilibrium need to be assessed thoroughly. These include the helix-forming tendencies of constituent amino acids, capping preferences at the C and N termini and preferences for the N1, N2, and N3 positions. Our helix-coil model N1N2N3 (Sun et al. 2000) takes all these factors into account. Briefly, every residue is considered to be in either a helix or coil conformation, giving a total of 2^N conformations for N residues. In each conformation, the N-cap residue has a weight n, followed by residue weights of n1, n2*w and n3*w. The C1 residue has a weight of c1, the C-cap c, and all helix interior residues from N4 to C2 have weights of w. Coil residues before and after the caps have weights of 1. Side-chain interactions between residues spaced i,i+3 and i,i+4 are also of considerable importance to helix stability, though irrelevant here. The statistical weight of each conformation is the product of the weights of each residue, taking into account the residue conformations. The sum of the statistical weights for every conformation is the partition function, and the population of each conformation is the statistical weight of the conformation divided by the partition function. All properties of the helix/coil equilibrium can be calculated from the populations and the partition function. Increasing the n1 value of a residue X will thus increase the population of all conformations where X is at the N1 position of a helix.

Design of the N1 peptide sequence
A residue at the N-terminal position in an acetylated peptide adopts a helical conformation when it has helical dihedral angles, and the acetyl CO group is hydrogen bonded to the peptide NH of the i+4 residue. This residue is bounded by an amide group on both sides, and the N-terminal acetyl group can act as an N-cap (Doig et al. 1994). N1 preferences can therefore be determined by substituting the first residue in an acetylated peptide and measuring the change in helix content.

Our peptide (CH₃CO-XA₄QA₄QA₂GY-NH₂) is intrinsically helical because of its high Ala content. X is the variable residue, and it affects helix content through both its N-cap (if X acts as the N-cap) and N1 (if acetyl is the N-cap) preferences. N-cap preferences (Serrano and Fersht 1989; Lecomte and Moore 1991; Bell et al. 1992; Serrano et al. 1992; Chakrabartty et al. 1993a; Forood et al. 1993; Lyu et al. 1993; Yumoto et al. 1993; Doig et al. 1994; Doig and Baldwin 1995) and preferences for the peptide interior (O'Neil and DeGrado 1990; Horovitz et al. 1992; Park et al. 1993; Blaber et al. 1994; Chakrabartty et al. 1994; Rohl et al. 1996; Myers et al. 1997; Yang et al. 1997; Pace and Scholtz 1998) have already been measured, leaving the N1 preference (n1 in helix-coil theory) values as the only unknown parameter. The N1 preference can thus be found by finding the value of n1 that gives predicted helix content in agreement with experiment. No side-chain interactions are present in this sequence. The energy scales of Rohl (Rohl et al. 1996) are used in the calculations, as they are complete and derived in similar peptide systems for the interior and N-cap positions. The Tyr residue is present to give the peptide a UV absorption for determination of concentration. The penultimate Gly ensures that the -helix almost always terminates at, or before, this position so that problems arising from a helical Tyr affecting the CD signal are minimized (Chakrabartty et al. 1993b). A peptide of this sequence has a helix content of 40% so that the CD signal is sensitive to a change in free energy of -helix formation. The design of the AQ peptide ensures that the contribution of the N1 residue to stability is not influenced by a charge on a free N terminus or by neighboring Gln residues included for solubility, as the Q residues are spaced i,i+5.

Helix contents of N1 peptides
The helix content of each peptide was measured by CD at 222 nm at a concentration of 10–30 µM in 10 mM NaCl, 5 mM sodium phosphate (pH 7.01) at 273 K. Measurements are made at 273 K because the helix-coil equilibrium favors the helical conformation at lower temperature and CD signals are consequently larger. CD measurements for these types of simple peptides are known to be proportional to concentration in this range (Stapley and Doig 1997) indicating that aggregation does not occur. The CD signal at 222 nm was converted to []₂₂₂ (see Materials and Methods), and the percentage helicity calculated with ±3% experimental error (Table 1).

It is tempting to draw conclusions about the observed stabilizing effects of residues at N1 on the basis of helix content relative to Ala. However, it is the underlying equilibrium constant for the helix-coil transition at N1, the n1 parameter, that defines the relative free energies for stabilization according to modern helix-coil theory. The stabilizing effects of the N-terminal residues are, therefore, described by both the n-cap and n1 energies, and helix-coil theory is able to separate the two parameters.

Determination of n1 values and G from helix contents
In all previous work, w, the equilibrium constant for adding a given residue to an -helix, has been determined assuming that the values of n1, n2, and n3 are no different from any other internal position. However, we have now examined the contributions of the N1 residues to stability in isolation from other interactions by considering the magnitude of n1 values specifically. n1 is formally defined as the equilibrium constant for the helix-coil transition at the N1 position (Sun et al. 2000). The percentage helicities measured for the N1 peptides have been converted to n1 for the helix-coil transition using a modified Lifson-Roig theory (see Materials and Methods). Initially, n1 for the four sequences with an X residue matching residues of the generic peptide (Ala, Gly, Gln, and Tyr) were solved simultaneously by fitting the equilibrium equation to the observed helicities using the N1N2N3 program (see Materials and Methods). Fitting a value for Tyr was problematic, as convergence to a positive value for the n1 parameter was not possible. It was, therefore, set to zero. This did not affect other n1 values significantly, as Tyr is found only at their C termini and, therefore, is unlikely to be in position N1 of an -helix in these peptides. Subsequently, these four n1 values were fixed for the calculation of n1 for the remaining peptides.

The residue n1 values are presented in rank order in Table 1. The order is different when compared with the order of helicity values. This indicates that the observed helicity depends on the relative magnitudes of the n1 parameter compared with the n-cap parameter and confirms that analysis using helix-coil theory is essential for understanding the equilibrium.

The n-cap and n1 parameters represent the probabilities that a particular residue will adopt either an N-cap conformation or an N1 conformation, respectively. When the n-cap value for an amino acid is large and of similar magnitude to the acetyl group (n-cap = 5.9; Doig et al. 1994), it will spend much of its time in a nonhelical N-cap conformation. Indeed, the large n-cap values available for Cys (5.4), Asn (6.8), and Tyr (4.9) tend to shift the equilibrium in favor of the N-cap state for the residue X. This competition between parameters results in low estimates for the n1 values.

All helix-coil parameters are interdependent, so the n1 values could be perturbed by errors in n values, in particular. This problem is most important when X in the sequence we used, CH₃CO-XAAAAQAAAAQAAGY-NH₂ has a high N-cap preference, such as Asp. In this case, the helices will often nucleate with X as the N-cap. When X has a low N-cap preference, the acetyl will usually be the N-cap, X will be at N1, and an error in n(X) will be small, as the conformation when X is the N-cap will be rarely populated. To investigate this problem, we recalculated n1 for Asp after recalculating the error range in n (found by varying the helix contents of the N-cap peptide [Doig and Baldwin 1995] by ±3% and refitting).

n(Asp) is in the range 4–7, and these values put n1(Asp) in the range 0.330 to 0.249 or ±0.07kcal/mol. Other amino acids are less sensitive to errors in n.

n1(Pro), for example, varies from only 0.086 to 0.111 (±0.07kcal/mol) if n(Pro) is varied from 1.35 to 0.1 (much larger than the real error range). Our results are therefore insensitive to errors in n-values and were indeed designed so that this is the case.

The N-cap energy of Cys⁰ has not been measured previously. To estimate n1(Cys), we set its n-cap parameter to that used for Ala; that is, 1. This yielded n1(Cys) = 0.04, which is comparable with that measured for a similar residue, Ser, n1 = 0.13, given the experimental uncertainty. However, calculations with the apparently more appropriate n-cap(Ser) value did not allow determination of n1(Cys) with a positive value. It is therefore likely that the real n-cap parameter for Cys in an uncharged environment is somewhere between that of Ala and Ser.

From the n1 values, the free energy change for the transfer of a residue from coil conformation to the N1 helical conformation, G, is given by -RT ln (n1). The free energy change relative to Ala, G_GXaa-GAla, was also calculated (Table 1). The data show that the N1 energies for the majority of the residues lie within 1 kcal mol^-1 of that measured for Ala. The hypothesis that stabilization of the -helix N terminus results from the presence of negatively charged amino acids is not universally supported by the G values measured in the AQ peptides. However, the neutral species of Asp and Glu are less stabilizing relative to their anionic forms.

Comparison with other reported n1 values
Recently, free energies for N1 have been reported for a series of AR peptides (Petukhov et al. 1998, 1999), and we have recently published a reanalysis of previously reported AK peptide data (Chakrabartty et al. 1993a) using the N1N2N3 program (Sun et al. 2000). The previously reported free energies for several N1 residues are very similar (Table 1), with Leu, Ser, Thr, Gln, Met, Ile, Val, Pro, and Gly having G 0.5 ± 0.1 kcal mol^-1. The one residue that does not follow this trend is Asn, which is most destabilizing at N1 for the short AK peptides.

The peptides studied here are 15 mers, with approximately double the helical content of those studied previously. These data, therefore, give more accurate N1 energies. Our new data indicate that the reported 1.4 kcal mol^-1 (Sun et al. 2000) is too low an estimate of n1 for Asn because we were unable to determine a positive value. This may be because the previously reported N-cap value (Doig and Baldwin 1995) used in the calculation is too high.

The G values presented in this study for Leu, Ser, Thr, Gln, Val, Pro, and Ile are similar (within error) compared to those described previously (Table 1). Proline destabilizes the -helix by 0.6 kcal mol^-1 relative to Ala, which is moderately high relative to the other 18 amino acid residues. Structurally, Pro is apparently well suited to the N1 position, as its dihedral angle is constrained to a helical conformation and it has no free peptide NH group. Interestingly, statistical preferences for Pro at N1 are the highest for any amino acid in both the global (compared to all protein positions) and local (compared to other helical positions) context, 2.6 and 5.8, respectively (Penel et al. 1999). This demonstrates that a high propensity does not necessarily indicate a large stabilization.

While propensities are measured relative to all other positions in a protein, N1 energies are measured relative to the coil state. The high propensity for Pro at N1 is thus likely to be because of the fact that Pro is highly destabilizing at most other locations within a protein, such as within an -helix or ß-sheet, rather than because of any intrinsic energetic preference for N1. The average global propensity for Pro in a type I turn is similarly high at 2.45 (Hutchinson and Thornton 1994). We suggest that this high propensity is, again, because Pro is tolerated at this location rather than because it stabilizes turns more than do other amino acids. The propensity measurements from statistics in protein structures cannot therefore substitute for free energy measurements. There are no other reported measurements of n1 preferences for Glu, Trp, His, Lys, Arg, Cys, Phe, or Tyr for comparison.

pH titrations
pH titrations were performed on those peptides with titratable groups to determine the pK_a values of the residue side chains in the N1 position (Fig. 1). The asymptotic values of ₂₂₂ for the different protonation states were used to fit helix-coil theory and, hence, find n1 values. The ellipticity data from the titrations were fitted to a Henderson-Hasselbach equation for two different pK_as, as all peptides contain Tyr at the C terminus. In general, we measured pK_a values within the broad range observed in proteins, that is, Asp/Glu, pK_a = 2–5.5; and His, pK_a = 5–8 (Fersht 1999). However, these ranges are too broad for comparisons with those pK_as measured here.

View larger version (52K): [in this window] [in a new window]   Fig. 1. pH titrations of CD signal at 222 nm for (a) N1(Asp), (b) N1(Glu), and (c) N1(His) peptides. N1-Xaa: CH3CO-XAAAAQAAAAQAAGY-NH2.

The pK_a for N1(Asp) (3.93 ± 0.03) is marginally lower than that seen in appropriate model compounds, and the pK_a of N1(Glu) is unperturbed (4.56 ± 0.09). The pK_a for N1(His) of 6.79 ± 0.03 is also normal given that the imidazole group has two microscopic dissociation constants, 6.5 and 7.1 (Kyte 1995).

In the AQ peptides, there can be no side chain–side chain interactions, so stabilization must come from other effects. The acetyl group is very probably hydrogen bonded to the NH group of the i+4 residue in a capping interaction and is not free to interact with the side chains at N1. In crystal structures of proteins, it is generally observed that the side chains of Asp and His are sterically constrained from forming favorable hydrogen bonds with the main chain (Penel et al. 1999) and, therefore, potentially interact solely with the macrodipole. However, an M2S1 hydrogen bond between the Glu side-chain carbonyl and the main chain of the N2 residue is possible, though energetically costly and unfavorable, as it is not in the preferred rotamer conformation (Penel et al. 1999). These facts support the hypothesis that the effect of N1 side-chain titration on -helix stability involves an interaction with the -helix macrodipole.

The free energy for protonation of Asp at N1 in the AQ peptide is 0.5 kcal mol^-1 (range of 0.3–0.6), and the anionic form is clearly more stable than the neutral species (Fig. 1a). Doig and Baldwin (1995) measured the free energy for protonation of Asp at the N-cap and reported a value 0.7 kcal mol^-1. The difference between these two values reflects the stronger interaction of the N-cap(Asp) with the free NH groups at the N terminus, as an N-cap Asp usually forms strong hydrogen bonds to the N2 and N3 NH groups (Doig et al. 1997). The protonated state of Glu was also found to be marginally disfavored at N1 by 0.2 kcal mol^-1 (range of 0.17–0.21 kcal mol^-1) and destabilization of the -helix at low pH is clearly observed in Figure 1b. Hence, negatively charged side chains stabilize the -helix compared with the neutral species by virtue of their larger n1 parameter.

Asp^- at N1 may be more stabilizing to the -helix compared with Glu^- because of the differences in distance between the deprotonated side chain and the macrodipole field and not hydrogen bonding per se. Similarly His⁺ may be more destabilizing than Lys⁺ and Arg⁺, as its charge is closer to the helix terminus.

The free energy for protonation of the His side chain is not determined, as an n1 value for His⁺ could not be estimated. However, titration of the N1(His) peptide to pH 12 demonstrates that the -helix is progressively stabilized from pH 5–8 and destabilized with ionization of the Tyr–OH between pH 8 and 12 (Fig. 1c). From this, we were able to measure the pK_a of the C-terminal Tyr to be 10.60 ± 0.13. The pK_a of Tyr is consistently measured to be 9.8 in a range of model compounds (Kyte 1995). The increased pK_a of Tyr may result from an unfavorable electrostatic interaction with the C-terminal dipole or partial negative charges on the terminal CO groups.

Other studies on alanine-based helical peptides have not reported the pK_a of the C-terminal Tyr. We believe that -helix destabilization between pH 9 and 12 was present in a previous study (Doig and Baldwin 1995), but the Tyr titration was obscured by the effect of three Lys residues included for solubility. The pK_as of peptides containing Arg, Lys, Cys, and Tyr side chains at N1 were not evaluated, as their measurements are complicated by overlapping titration of Tyr at the C terminus.

Direct observation of the -helix macrodipole is not possible, and its existence is inferred by the observation that charged side chains and other ionic species are commonly found near -helix termini in crystal structures. The pK_as of the side chains at the N1 position of the AQ -helix are not significantly perturbed by a macrodipole interaction. Stabilization of the -helix on ionization of the acidic side chains is indisputable, and protonation of His is so unfavorable that we cannot measure this n1. The helicity of these peptides is very sensitive to interactions at the N1 position such that a small interaction of an anionic side chain with the dipole leads to measurable changes in []₂₂₂ (but no detectable changes in pK_a). It is also possible that the coil form of the polypeptide is destabilized by the anionic side chain or stabilized by the protonated form in any number of possible interactions with the rest of the molecule.

Correlation of n1 energies with N1 propensities and w values
We recently analyzed the local and global propensities for every amino acid in first turn of the -helix (Penel et al. 1999). The global propensity value is an indicator of the abundance of a particular amino acid in the N1 position relative to the whole molecule. The relationship between the observed frequency of a particular residue at N1 and the experimentally determined n1 values presented in this report has been investigated (Fig. 2). A correlation between the two parameters (R = 0.49) is clearly observed, with Pro as an outlier (see above). Thus, the energy cost for placing a residue at N1 in our model peptide is a reasonable reflection of the process in natural proteins. The fact that the correlation is not perfect shows the necessity of measuring free energies rather than solely determining propensities from crystal structures. Ala has a high N1 preference, while that for Gly is low, as in -helix interior positions. This is presumably because the flexible backbone of Gly loses more backbone conformational entropy when it folds than for other amino acids.

View larger version (20K): [in this window] [in a new window]   Fig. 2. The relationship between n1 and N1 residue propensity. The N1 propensities (the observed relative abundance in crystal structures) are taken from Penel et al. (1999). Proline was not included in the regression analysis because it has an abnormally high propensity for N1 (see text).

View larger version (20K): [in this window] [in a new window]   Fig. 3. The relationship between w and n1 in the -helix. The n1 values are calculated from helix-coil theory as described herein. The w values are taken from Rohl and Baldwin (1996).

	Conclusion

TOP Abstract Introduction Results and discussion Conclusion Materials and methods References

	Materials and methods

TOP Abstract Introduction Results and discussion Conclusion Materials and methods References

 
Peptide synthesis
Peptides were synthesized by the solid-phase method. C-terminal amides were made using Rink Amide resin (CN Biosciences). N termini were acetylated with acetic anhydride/pyridine. Peptides were cleaved from the resin using 95% trifluoroacetic acid, 2.5% triisopropylsilane, 2.5% water. Peptides were purified by C₁₈ reverse phase HPLC, and their molecular weights were checked by ES or MALDI mass spectrometry. Peptide purity was checked by analytical C₁₈ HPLC. Peptide concentration was determined by measuring tyrosine UV absorbance of aliquots of stock solution dissolved in water or 6 M guanidine hydrochloride using ₂₇₅ = 1390 M^-1 cm^-1 or ₂₇₅ = 1450 M^-1 cm^-1, respectively (Brandts and Kaplan 1973). Concentrations of peptides containing Trp were determined in 6 M GuHCl (pH 7.0), 25°C using ₂₈₁ (Trp) = 5690 M^-1 cm^-1 + ₂₈₁ (Tyr) = 1250 M^-1 cm^-1 (Edelhoch 1967).

Circular dichroism measurements
Equilibrium CD measurements were made at 222 nm with a Jasco J810 spectropolarimeter at 273 K. Ellipticity was measured in 10 mM NaCl, 5 mM sodium phosphate (pH 7.0) in a quartz cell with a 0.1 cm path length. The Cys peptide was measured in the presence of 1 mM dithiothreitol. CD data in millidegrees was converted to mean residue ellipticity using []₂₂₂ = /(molar concentration x 15 residues), in deg cm² dmol^-1. Helix content was calculated as []_{222(observed)}/[]_222(max). []_222(max) is given by -40,000(1–2.5/n), where n is the number of amino acids in the peptide (Chakrabartty et al. 1991).

pH titrations were performed in a 1.0-cm quartz cell at 273 K. The buffer used for pH titrations contained 10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate, and 1 mM sodium citrate. The pH meter was calibrated for measurements at 273 K. The pH was adjusted during the titrations with aliquots of HCl or NaOH. Data was only used in the pH range where the equilibrium is reversible (thus ruling out deamidation or other irreversible effects as the cause of the pH changes). The ellipticity data from the titrations were fitted to a Henderson-Hasselbach equation for two different pK_as

where, pK_a1 and pK_a2 are the pK_as measured for the acid– base equilibrium at low and high pH, respectively. []_{222 low_pH}, []_{222 mid_pH}, and []_{222 high_pH} are the molar ellipticities measured at 222 nm at the titration end points at low, mid, and high pH. []_{222 mid_pH–high_pH} is the change in molar ellipticity associated with pK_a2. The free energies for protonation were calculated using G = -RT ln[n1(charged)/n1(uncharged)].

Determination of helix-coil parameters
n1 values were determined using a statistical mechanics algorithm implemented in the program N1N2N3 (http://www.bi.umist.ac.uk/users/mjfajdg/n1n2n3.htm). N1N2N3 implements the modified Lifson-Roig helix-coil theory (Sun et al. 2000) to calculate the helix content of a given peptide sequence. The inputs to the program are the peptide sequences and corresponding experimental helix contents; a library of w, n-cap, n1, n2, n3, c1, and c-cap values for each amino acid; an n-cap value for acetyl and a c-cap value for amide; and a list of parameters to be determined. All n2, n3, and c values were set to 1. Parameters were varied until they converged on essentially unchanging values.

	Acknowledgments

 
The Michael Barber Mass Spectrometry Facility at UMIST is thanked for verifying peptide identity. This work was supported by the Biotechnology and Biological Sciences Research Council (Grant 36/B09794).

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.

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