Structural and thermodynamic consequences of introducing {alpha}-aminoisobutyric acid in the S peptide of ribonuclease S

Girish S. Ratnaparkhi1,2, Satish Kumar Awasthi1,3, P. Rani1, P. Balaram1,4,4 and R. Varadarajan51,4,4

1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 and 4 Chemical Biology Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, 560 004, India


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The S protein–S peptide interaction is a model system to study binding thermodynamics in proteins. We substituted alanine at position 4 in S peptide by {alpha}-aminoisobutyric acid (Aib) to investigate the effect of this substitution on the conformation of free S peptide and on its binding to S protein. The thermodynamic consequences of this replacement were studied using isothermal titration calorimetry. The structures of the free and complexed peptides were studied using circular dichroic spectroscopy and X-ray crystallography, respectively. The alanine4Aib replacement stabilizes the free S peptide helix and does not perturb the tertiary structure of RNase S. Surprisingly, and in contrast to the wild-type S peptide, the {Delta}G° of binding of peptide to S pro, over the temperature range 5–30°C, is virtually independent of temperature. At 25°C, the {Delta}{Delta}G°, {Delta}{Delta}H°, {Delta}{Delta}S and {Delta}{Delta}Cp of binding are 0.7 kcal/mol, 2.8 kcal/mol, 6 kcal/mol.K and –60 kcal/mol.K, respectively. The positive value of {Delta}{Delta}S is probably due to a decrease in the entropy of uncomplexed alanine4Aib relative to the wild-type peptide. The positive value of {Delta}{Delta}H° is unexpected and is probably due to favorable interactions formed in uncomplexed alanine4Aib. This study addresses the thermodynamic and structural consequences of a replacement of alanine by Aib both in the unfolded and complexed states in proteins.

Keywords: {alpha}-aminoisobutyric acid crystal structure/conformational entropy/RNase S/titration calorimetry


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bovine pancreatic ribonuclease A (RNase A) may be cleaved with subtilisin to give two fragments, S peptide (S pep; residues 1–20) and S protein (S pro; residues 21–124) (Richards and Vithyathil, 1959). These fragments can be reconstituted to give the catalytically active complex ribonuclease S (RNase S), having a structure very similar to that of RNase A (Kim et al., 1992Go). As residues 16–20 are not important for binding to S pro, we use a truncated, synthesized version of S pep in order to study the S pep–S pro interaction. Amino acids, including those with unusual side chains, can be substituted into S pep during synthesis. In earlier studies aimed at understanding the thermodynamic stability of proteins, nine hydrophobic residues ranging in size from Gly to Phe were substituted for the buried Met13 using peptide synthesis. The free energies ({Delta}G°), enthalpies ({Delta}H°), entropies ({Delta}S) (Connelly et al., 1990Go) and the heat capacities ({Delta}Cp) (Varadarajan et al., 1992Go; Thomson et al., 1994Go) of the binding of these substituted analogs to S pro were measured using titration calorimetry. The studies showed significant changes in these binding parameters as a result of the substitutions. All nine analogs were crystallized as a complex with S pro and the structures solved (Varadarajan and Richards, 1992Go; Thomson et al., 1994Go). An attempt was made to interpret the thermodynamic changes based on the structural data. The thermodynamic changes showed large and compensating changes in {Delta}{Delta}H° (25°C) and {Delta}{Delta}S (25°C) (Varadarajan and Richards, 1992Go; Thomson et al., 1994Go) in several mutants.

Using an alternative approach, we have attempted to alter the conformation of the free S pep without substituting any residue involved in binding by introducing the unusual amino acid residue {alpha}-aminoisobutyric acid (Aib) (Balaram, 1992Go) in the S pep. Aib residues are known to constrain conformationally polypeptide chains and stabilize {alpha}-helical and turn conformations (Prasad and Balaram, 1984Go). Aib residues are non-standard amino acids found in microbial membrane channel forming peptides and have significant potential in protein engineering (Balaram, 1992Go). Data on the structural and thermodynamic consequences of Aib replacements in proteins are scarce, especially in protein–protein interactions. Introduction of an Aib in the S pep of RNase S allows us to study the effect of an Aib replacement on the structure of unfolded S pep, folded RNase S and on the S pep–S pro interaction. Residues 3–13 form an {alpha}-helix in RNase S. It is therefore expected that the substitution of alanine (A) by Aib in S pep will increase the binding affinity of S pep for S pro by decreasing the conformational entropy of free S pep. We have chosen to replace alanine at position 4 by Aib (A4Aib). Residue 4 was chosen as a site for replacement as it is not a buried residue, does not interact significantly with S pro and is not involved in intermolecular packing with other molecules in the crystal structure of RNase S (Kim et al., 1992Go). Modelling studies showed that substitution of the hydrogen by a methyl group at the C{alpha} position of Ala 4 did not result in any unfavourable steric overlap. Thus Aib at position 4 in the S pep should only affect the binding of S pep to S pro by perturbing the conformation of the free S pep without affecting any of the residues packing against S pro.

The free state of the A4Aib peptide was characterized using circular dichroic (CD) spectroscopy and the complex of A4Aib with S pro, hereafter referred to as RNase S (A4Aib), by X-ray crystallography. The enthalpy, entropy, free energy and heat capacity of A4Aib binding to S pro were measured using isothermal titration calorimetry. In addition, the thermal stability of the complex was characterized by CD measurements. These studies allowed us to characterize completely the effects of changes in the conformation of S pep on the stability of RNase S.

Understanding the structural and thermodynamic consequences of Ala to Aib replacements on both folded and unfolded states of polypeptide chains is essential as these replacements are increasingly being made to increase protein stability (de Filippis et al., 1998Go), improve antigen display (Sakarellos-Daitsiotis et al., 1999Go) and in drug design (Hewage et al., 1999Go; Howl et al., 1999Go). Aib has recently been used to improve protein–protein recognition in biologically relevant molecules such as the gp120 coat protein of HIV (Stanfield et al, 1999Go) and for de novo protein design (Karle et al., 2000Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
S pro and S pep

RNase S was made from RNase A (Sigma, Type XII A), by subtilisin cleavage (Richards and Vithyathil, 1959). The S pro fragment was separated from the S pep by gel filtration under denaturing conditions and further purified by ion-exchange chromatography as described (Nadig et al., 1996Go) and stored in water at –20°C. The purified S pro gave a single band on a silver-stained SDS–PAGE gel and also gave a single peak at the expected molecular weight of 11 527.8 on a mass spectrometer. The S pro concentration was based on a molar absorptivity of 9.56 mM–1 cm–1 at 280 nm (Connelly et al., 1990Go).

The control (S16) peptide used for the study has the sequence KETAAAKFERQHLDSG and is identical with the S15 (M13L) peptide used in earlier studies (Varadarajan and Richards, 1992Go), except that the C-terminus has an extra glycine residue, rather than being amidated. This difference was because of the non-availabiity of the appropriate resin to make the amidated peptide. The tendency of methionine to oxidize and modify the binding properties of S pep is removed in the M13L peptide. In the A4Aib peptide, alanine 4 in S16 peptide is replaced by {alpha}-aminoisobutyric acid. This S16 and the A4Aib peptides were synthesized by stepwise solid-phase synthesis (Atherton and Sheppard, 1989Go) using N-fluorenylmethoxycarbonyl (Fmoc) polyamide active chemistry on Ultrasyn A resin (Novabiochem). The peptides were synthesized on a 0.1 mmol scale. The Fmoc group was removed from the N-terminus by using 20% piperidine in DMF for 15 min in a continuous flow system. The resin was removed from the column, washed with diethyl ether and dried in vacuo. TFA–anisole–EDT (95:5:1) was found to give complete cleavage of the peptide from the resin after 10 h in an inert atmosphere. The TFA was evaporated and the peptide was precipitated in diethyl ether and further washed with diethyl ether repeatedly to remove the scavenging agents. The peptides were purified by reversed-phase chromatography using a Vydac C18 preparative column on a Waters HPLC system with water–acetonitrile containing 0.1% TFA as eluent. The S16 peptide gave a single major peak whereas the A4Aib peptide gave two peaks during purification, which were separated in this step. Electrospray mass spectrometry was used to confirm the mass of the control peptide (1786.0 Da) and also to identify the correct peak in case of the A4Aib peptide (1802.0 Da). The other peak was identified as that of the A4Aib peptide with a deletion of threonine at position 3 (1527 Da).

CD spectroscopy

The CD data were collected on a JASCO J715 spectrometer interfaced to a Neslab RTE-110 circulating water-bath. The temperature melts were monitored at a single wavelength (222 nm) for both the S pep and RNase S melts. The solution conditions were 5 mM sodium acetate and 10 mM NaCl, pH 6. The concentrations used for the peptide alone were 40 µM with a pathlength of 2 mm. The melts for the RNase S complex were done by mixing 30 µM S pro with a molar excess (1.3) of S pep (40 µM). The RNase S melts were also done under conditions where the binding affinity of S pep to S pro was enhanced, i.e. 15 mM sodium phosphate, pH 7.

Titration calorimetry

The calorimetric experiments were done on an isothermal titration calorimeter from MicroCal. The titrations were performed in the temperature range 6–30°C as previously described (Connelly et al., 1990Go; Varadarajan et al., 1992Go; Thomson et al., 1994Go). Data were analyzed using the MCS-ORIGIN software package, assuming a single set of identical binding sites. Figure 1Go shows a representative trace of a titration of the A4Aib peptide with S pro at 25°C, pH 6.0, 0.1 M NaCl, 0.05 M sodium acetate buffer. The convention {Delta}{Delta}X° (where X is G, H, S or Cp) = {Delta}X°mutant {Delta}wild-type has been followed throughout (Connelly et al., 1990Go).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1. Representative isothermal titration calorimetric profile of the binding of the A4Aib peptide to S pro at 25°C. The data were fitted (solid line) to a single-site binding model to obtain the {Delta}H° and K values using the ORIGIN package.

 
Crystallization, data collection and reduction

Crystallization was carried out as described (Varadarajan and Richards, 1992Go). The final conditions in the drop were 4–12 mg/ml protein, 3 M CsCl, 0.1 M acetate buffer, pH 5.75 and 35% (NH4)2SO4. The crystals were stabilized in 75% (NH4)2SO4, pH 4.75 and the CsCl was washed out by repeated buffer changes. The crystals were stored in this solution for 1–8 weeks. The size of the crystals was of the order of 0.5x0.5x0.3 mm. X-ray diffraction data were collected for a single crystal at 20°C using a MAR imaging plate detector system mounted on a Rigaku RU200 rotating anode operating at 40 kV, 60 mA. The dataset was collected by recording 1.0° oscillation images. Diffraction data were recorded over an oscillation range of 150°. Each frame was collected for 200–300 s. Table IGo lists the RNase S (A4Aib) data set collected along with the control data set. The control structure was the RNase S (M13L) structure solved by Varadarajan and Richards (1992). Data were reduced using the MAR-XDS suite of programs (Kabsch, 1993Go).


View this table:
[in this window]
[in a new window]
 
Table I. Summary of data collection, reduction and refinement for the RNase A4Aib and its control structure
 
Refinement and analysis

Refinement was carried out using simulated annealing (SA) with the slow cooling protocol of XPLOR (Brunger, 1996Go) as described by Ratnaparkhi and Varadarajan (1999), with the additional calculation of Rfree (Brunger, 1992Go; Kleywegt and Brunger, 1996Go). Manual model building was done against {sigma}-a weighted (Read, 1986Go) 2Fo Fc and FoFc difference Fourier maps, using FRODO (Jones, 1985Go). The statistics for the final refined structures are given in Table IGo. The XPLOR parameter and topology files for the Aib residue were taken from the Brunger laboratory web site (http://atb.csb.yale.edu/hetero).

The structures were analyzed using root mean square deviation (r.m.s.d.) and {Delta}B-factor plots per residue for MC and SC. Parameters such as accessibility (Connolly, 1983Go) and depth (Chakravarty and Varadarajan, 1999Go) were examined. {sigma}-A weighted omit maps (Read, 1986Go; Brunger, 1996Go) were calculated to confirm structural changes and these regions were rebuilt based on the 2Fo Fc and FoFc omit maps.

Quality of the structure

The final, refined structures show excellent stereochemistry (Table IGo) with no violations for bonds, angles, dihedrals or impropers based on the default threshold values in XPLOR. The structures had reasonable R and Rfree values (Table IGo). The quality of the structures was checked using the program PROCHECK (Laskowski et al., 1993Go) and the structure showed good stereochemistry when compared with the database of high-resolution structures with a g-factor of 0.15. The structure showed {phi}, values in the core and allowed regions of the Ramachandran plot.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Conformation of the A4Aib peptide in solution

The far-UV CD profiles of the S16 and A4Aib peptide in solution were similar to those of the S15 peptides (data not shown) in the temperature range 3.3–35°C (Connelly et al., 1990Go). Both the S16 peptides were random coils at 25°C and showed an increase in helicity at lower temperatures like other S pep analogs (Mitchinson and Baldwin, 1986Go; Connelly et al., 1990Go). The temperature dependence of the CD signal (monitored at 222 nm) of the A4Aib peptide is indicative of a conformational transition, unlike that of the control peptide (Figure 2AGo). At 3.3°C, pH 6 the A4Aib peptide shows higher ellipticity (222 nm) than the control peptide, indicating that the S pep helix has been stabilized to a significant extent at lower temperatures. The fraction helicity (f) of the peptides can be calculated as described (Goldberg and Baldwin, 1998Go). The f value for the Ala4Aib peptide was 15% compared with 5% for the S16 peptide. The ribonuclease S15 peptide, which is similar to the S16 peptide, in comparison has f = 7% at 3°C, pH 5.3 (Mitchinson and Baldwin, 1986Go).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. (A) Mean residue ellipticity ([{theta}]222) of the A4Aib (•) and control ({circ}) peptides as a function of temperature. (B) Temperature dependence of {Delta}H° of the binding of the A4Aib(•) and the control ({circ}) peptide to S pro. The {Delta}H°s are fitted to a straight line in the temperature range 6–25°C to derive the {Delta}Cp of binding.

 
Binding of S16 and A4Aib to S pro

Titration calorimetry has been used to characterize completely the binding thermodynamics of the S16 and the A4Aib peptides to S pro (Figure 1Go) under the same solution conditions as used for earlier studies (Connelly et al., 1990Go).

The A4Aib peptide binding to S pro is slightly weaker at all temperatures investigated by titration calorimetry (Table IIGo) relative to its control. A surprising feature of the A4Aib binding thermodynamics is that the binding affinity is practically independent of temperature. The binding constant (K) decreases by a factor of three over the temperature range 6.0–35.0°C for the A4Aib binding to S pro. In contrast, for S16 there is a 20-fold decrease in K over the same temperature range (Table IIGo). The behavior of the A4Aib peptide is unusual because the S15 peptide along with the eight mutants studied earlier all showed a large decrease in K (50–100-fold) over this temperature range. Whereas the magnitude of {Delta}G° decreases with temperature for all peptides previously studied, for A4Aib the magnitude actually increases slightly with increasing temperature. The enthalpy ({Delta}H°) of binding of A4Aib is slightly lower than that of the control peptide at all temperatures investigated (Figure 2BGo). The heat capacities ({Delta}Cp) of binding of the S16 and the A4Aib peptides are not very different and compare well with those of other mutant S15 peptides (Varadarajan et al., 1992Go). At 25°C (Table IIIGo) the thermodynamic parameters of S16 and Aib binding to S pro are similar. The {Delta}{Delta}G°, {Delta}{Delta}H°, {Delta}{Delta}S and {Delta}{Delta}Cp of binding are 0.7 kcal/mol, 2.8 kcal/mol, 6 kcal/mol.K and –60 kcal/mol.K, respectively.


View this table:
[in this window]
[in a new window]
 
Table II. Thermodynamic characterization of the control (S16) and A4Aib binding to S pro (6–35°C)
 

View this table:
[in this window]
[in a new window]
 
Table III. Standard enthalpy and heat capacity for S pep binding to S pro (25°C)
 
The temperature dependence of {Delta}G° (Table IIGo) indicates that at higher temperatures (>40°C), the RNase S (Ala4aib) complex may be more stable than the RNase S16 structure. We could not collect titration calorimetric data at higher temperatures because of aggregation of S pro at temperatures >=40°C. In order to test whether the A4aib mutation made RNase S more stable to thermal denaturation, temperature melts of RNase S (A4Aib) and RNase S16 were done at a protein concentration (30 µM) similar to the concentration of RNase S used for the titrations. The melts were done both in 5 mM sodium acetate, 10 mM NaCl, pH 5 (data not shown) and also under conditions where the S pro–S pep interaction is enhanced (15 mM sodium phosphate, pH 7). Figure 3Go shows that replacing A4 with Aib does not perturb the temperature melting profile of RNase S. Tertiary CD spectra (data not shown) of RNase S (A4Aib) and RNase S16 are identical, indicating that both structures had similar tertiary structure and similar overall stability. The three-dimensional structure of RNase S (A4Aib) was solved in order to confirm the similarity in the tertiary structure.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Temperature melt of RNase S16 and RNase S (A4Aib) in 15 mM sodium phosphate, pH 7. Symbols as in Figure 2Go.

 
Crystal structure of the RNase S (A4Aib) mutant

The RNase S (A4Aib) complex crystallizes in similar conditions to RNase S15 and has similar cell parameters (Table IGo). The electron density for Gly16 is not visible and therefore it has been deleted from the model. This is not surprising as residues 16–23 are also disordered in the structure of the 20-residue S pep complexed to S protein (Kim et al., 1992Go). The structure of the control RNase S16 was not solved and its structure was assumed to be identical with that of RNase S15 (M13L) (Varadarajan and Richards, 1992Go). The RNase S (A4Aib) complex, in solution, was as active as RNase S16 or RNase S15 as measured by the 2',3'-cyclic CMP assay (Crook et al., 1960Go), indicating that the tertiary structure is not perturbed by the substitution. R.m.s.d. plots indicate that the introduction of Aib at position 4 does not affect the structure of RNase S both overall as well as near the mutation site (Figure 4AGo). {Delta}B-factor plots also do not show significant changes (Figure 4BGo). The replacement of Ala by Aib is confirmed by 2FoFc and FoFc electron density and the presence of density in omit maps (Read, 1986Go; Brunger, 1996Go). Figure 5AGo shows the Aib side chain (ball-and-stick) in the RNase S (A4Aib) structure. Figure 5BGo shows the Aib side chain positioned in the 2FoFc density along with some neighboring residues. The Aib main chain has a conformation similar to that of the Ala residue in the wild-type structure. The addition of a CH3 group is accomplished without perturbing nearby residues. This is because the Aib Cß2 atom is solvent exposed and occupies space not occupied by any protein or water atom. There are no symmetry-related groups in the vicinity of residue 4 and therefore the packing of the molecules in the crystal is not perturbed.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. (A) R.m.s.d. plot. MC and SC r.m.s.d.s are represented by continuous and dashed lines, respectively. The large side chain r.m.s.d.s are generally due to lack of density for side chains on the surface. The structure was superposed on its control structure before calculating the r.m.s.d. (B) {Delta}B-factor plot. The restrained B-factor per residue for the control structure was subtracted from the corresponding B-factor of the control structure to obtain the {Delta}B-factor plot. The filled bars indicate the MC {Delta}B-factors and the lines represent the SC {Delta}B-factors. The secondary structure and accessibility representation on top of the panel is an output of the program PROCHECK (Laskowski et al., 1993Go).

 



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 5. (A) MOLSCRIPT (Kraulis, 1991) representation of the RNase S (A4Aib) structure. The Aib4 C{alpha} atom and the side chain are shown in ball-and-stick format. (B) Stereo plot showing the superposed structure of the RNase S (A4Aib) structure (thick line) over that of the control RNase S (M13L) structure (thin line) in the region of residue 4. A 2FoFc electron density map (contoured at 1{sigma}) for the Aib residue is also shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
{alpha}-Aminoisobutyric acid (Aib) belongs to the class of {alpha},{alpha}-disubstituted amino acids. These amino acids have been introduced in peptides to stabilize secondary structure elements such as helices and turns, and also in protein design (Prasad and Balaram, 1984Go; Balaram, 1992Go). {alpha},{alpha}-Disubstituted amino acids are naturally present in microbial peptide antibiotics, several of which have been reported to form transmembrane ion channels. Aib and (R)-(–)-2-amino-2-methylbutyric acid (D-Iva) are the most common of this class of amino acids. A large body of work has been done on the effect of non-proteinogenic amino acids on peptide conformation (Prasad and Balaram, 1984Go). The presence of the geminal methyl groups at the C{alpha} position greatly reduces the sterically allowed regions of conformational space for the peptide backbone. The lowest energy corresponds to the helical region of the Ramachandran map ({phi} = ±57 ± 20, {phi} = ±30 ± 20). Crystallographic and NMR data collected on peptides containing Aib show that, with the exception of small peptides incapable of forming intermolecular hydrogen bonds or in cyclic systems, almost all other residues cluster around the left- or right-handed helical regions. Thus the presence of Aib stabilizes helices or turns in peptides. Aib is increasingly being introduced in larger polypeptides and proteins to improve stability (De Filippis et al., 1998Go) or modify conformational properties (Ghiara et al., 1997Go). Aib has recently been introduced (Aib142) into the V3 loop of HIV-1 with the intent of ordering it in a conformation similar to its Fab bound state and further use this strategy to mimic biologically relevant structural forms of the V3 loop (Ghiara et al., 1997Go; Stanfield et al., 1999Go). It is therefore essential that the thermodynamic and structural consequences of introducing Aib be understood.

We introduced Aib in a fragment complex, RNase S, to study the effect of the introduction of Aib on the conformation of the free S pep (a model for the unfolded state; Mitchinson and Baldwin, 1986Go), the binding of S pep to Spro (a model for protein–protein interaction), and the stability of the final folded RNase S (the folded state). RNase S is an ideal system to introduce non-natural amino acids into a protein as synthetic S peps with non-natural amino acids can bind to S pro. The binding thermodynamics of S pep analogs to S pro gives us an insight into the folding and stability of proteins. The RNase S complex is easily crystallized (Varadarajan and Richards, 1992Go), allowing the study of the structural perturbations caused by the introduction of Aib in the tertiary structure of the protein. Ala4 is part of the 3–13 helix of the S pep and the substitution was predicted to stabilize the S pep helix in solution.

The introduction of Aib leads to an increase in helicity at low temperatures (Figure 2AGo). This indicates that Aib stabilizes the S pep helix marginally at lower temperatures. However, at room temperature, both the S16 and A4Aib peptides appear to be unfolded. Hence the observed binding thermodynamics represents the binding of largely unstructured S pep to S pro to give an RNase S complex in which the peptide forms an {alpha}-helix. The binding reaction can conceptually be broken up into two steps, first the transition of the free peptide from an unstructured to helical conformation and second the binding of helical peptide to S pro. At room temperature neither S16 nor A4Aib contains detectable amounts of helix by CD. However, the unfolded A4Aib will still have fewer accessible conformations than the corresponding unfolded S16. Consistent with this assertion, the {Delta}S of binding is more positive for A4Aib than for S16 (Table IIGo). However, the {Delta}H° for binding is also more positive for A4Aib. This is unlikely to be due to steric strain in the folded state as the substitution is made at a solvent-exposed location. The crystal structure confirms that there is enough space for the additional methyl group in Aib to be accommodated without movement of nearby residues. Since the site is solvent exposed, the observed changes are also unlikely to result from differences in the hydrophobic driving force for binding of the two peptides. One other possibility is that the positive value of {Delta}{Delta}H° may result from more favorable interactions in the unfolded state of the peptide that occur because this peptide is more ordered. The value of {Delta}{Delta}G° is about 1 kcal/mol at low temperature but decreases with increasing temperature and at 35°C both A4Aib and S16 have identical values of {Delta}G°. The decrease in magnitude of {Delta}G° with increasing temperature observed for S16 is expected and results from the unfavourable conformational entropy change upon binding. The magnitude of this conformational entropy contribution to {Delta}G° increases with temperature. The surprising lack of temperature dependence of {Delta}G° for A4Aib is likely to be due, in part, to the reduced conformational entropy of the uncomplexed peptide as a result of introduction of the Aib residue. The present work shows that it possible to introduce Aib at a solvent-exposed site without affecting the structure of the protein. Although Aib reduces the conformational entropy of the unbound state, there is no increase in protein stability, because of significant enthalpy–entropy compensation. This study shows that the effects of Aib on protein folding thermodynamics are hard to predict even in cases where it can be introduced without unfavorable steric interactions.


    Notes
 
2 Present address: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560 065, India Back

3 Present address: Laboratory of Medical Biochemistry B, Department of Medical Biochemistry and Genetics, The Panum Institute, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark Back

4 To whom correspondence should be addressed. E-mail: varadar{at}mbu.iisc.ernet.in Back


    Acknowledgments
 
We thank Professor Jayant Udgaonkar, National Centre for Biological Sciences, Tata Institute for Fundamental Research, Bangalore, for the use of the titration calorimeter. Computational facilities of the Supercomputer Education Research Center and the Interactive Graphics facility, Indian Institute of Science, were utilized for this work. The X-ray data were collected at the Image Plate Facility at the Molecular Biophysics Unit. This work was supported by grants from DST and DBT, Government of India, to R.V.

The crystal structure and structure factors of the ribonuclease S (A4Aib) structure have been deposited at and released by the RCSB protein data bank as entry 1FEV.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Atherton,E. and Sheppard,R.C. (1989) In Rickwood,D., Hames,B.D. (eds), Solid Phase Peptide Synthesis. A Practical Approach. IRL Press.

Balaram,P. (1992) Curr. Opin. Struct. Biol., 2, 845–851.

Brunger,A.T. (1992) Nature, 355, 472–474.[ISI]

Brunger,A.T. (1996) X-PLOR Version 3.851. Yale University, New Haven, CT.

Connelly,P.R., Varadarajan,R., Sturtevant,J.M. and Richards,F.M. (1990) Biochemistry, 29, 6108–6114.[ISI][Medline]

Connolly,M.L. (1983) Science, 221, 709–713.[ISI][Medline]

Chakravarty,S. and Varadarajan,R. (1999) Struct. Fold. Des., 7, 723–732.[Medline]

Crook,E.M., Mathias,A.P. and Rabin,B.R. (1960) Biochem J., 74, 234–238.[ISI][Medline]

de Filippis,V., De Antoni,F., de Laurento,P.P. and Fontana,A. (1998) Biochemistry, 37, 1686–1696.[ISI]

Ghiara,J.B., Ferguson,D.C., Satterwait,A.C., Dyson,H.J. and Wilson,I. (1997) J. Mol. Biol., 266, 31–39.[ISI][Medline]

Goldberg,J.M. and Baldwin,R.L. (1998) Biochemistry, 37, 2556–2563.[ISI][Medline]

Hewage,C.M., Jiang,L., Parkinson. J.A., Ramage,R. and Sadler. I.H. (1999) J. Pept. Res., 53, 223–233.[ISI][Medline]

Howl,J., Prochazka,Z., Wheatley,M. and Slaninova,J. (1999) Br. J. Pharmacol., 128, 647–652.[Abstract/Free Full Text]

Jones,T.A. (1985) Methods Enzymol., 115, 157–171.[ISI][Medline]

Kabsch,W. (1993) J. Appl. Crystallogr., 26, 795–800.[ISI]

Karle,I.L., Das,C. and Balaram,P. (2000) Proc. Natl Acad. Sci. USA, 97, 3034–3037.[Abstract/Free Full Text]

Kim,E.E., Varadarajan,R., Wyckoff,H.W. and Richards,F.M. (1992) Biochemistry, 31, 12304–12314.[ISI][Medline]

Kleywegt,G.J. and Brunger,A.T. (1996) Structure, 4, 897–904.[ISI][Medline]

Kraulis,PJ. (1991) J. Appl. Crystallogr. 24, 946–950.

Laskowski,R.A., MacArthur,M.W., Moss,D.S. and Thornton,J.M. (1993) J. Appl. Crystallogr., 26, 283–291.[ISI]

Mitchinson, C. and Baldwin,R.L. (1986) Proteins, 1, 23–33.[Medline]

Nadig,G., Ratnaparkhi,G.S., Varadarajan,R. and Vishveshwara,S. (1996) Protein Sci., 5, 2104–2114.[Abstract/Free Full Text]

Prasad,B.V.V. and Balaram,P. (1984) CRC Crit. Rev. Biochem., 16, 307–345.[ISI][Medline]

Read,R.J. (1986) Acta Crystallogr., A42, 140–149.[ISI]

Ratnaparkhi,G.S. and Varadarajan,R. (1999) Proteins, 36, 282–294.[ISI][Medline]

Richards,F.M. and Vithayathil,P.J. (1959) J. Biol. Chem., 234, 1459–1464.[Free Full Text]

Sakarellos-Daitsiotis,M., Tsikaris,V., Sakarellos,C., Vlachoyiannopoulos,P.G., Tzioufas,A.G. and Moutsopoulos,H.M. (1999) Vaccine, 18, 302–310.[ISI][Medline]

Stanfield,R.L., Cabezas,A.C., Satterthwait,A.C., Stura,E.A. and Profy,A.T. (1999) Structure, 7, 131–142.[ISI][Medline]

Thomson,J., Ratnaparkhi,G.S., Varadarajan,R., Sturtevant,J.M. and Richards,F.M. (1994) Biochemistry, 33, 8587–8593.[ISI][Medline]

Varadarajan,R. and Richards,F.M. (1992) Biochemistry, 31, 12313–12327.

Varadarajan,R., Connelly,P.R., Sturtevant,J.M. and Richards,F.M. (1992) Biochemistry, 31, 1421–1426.[ISI][Medline]

Received March 2, 2000; revised August 8, 2000; accepted September 14, 2000.