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
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Abstract |
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Keywords: -aminoisobutyric acid crystal structure/conformational entropy/RNase S/titration calorimetry
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Introduction |
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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 -aminoisobutyric acid (Aib) (Balaram, 1992
) in the S pep. Aib residues are known to constrain conformationally polypeptide chains and stabilize
-helical and turn conformations (Prasad and Balaram, 1984
). Aib residues are non-standard amino acids found in microbial membrane channel forming peptides and have significant potential in protein engineering (Balaram, 1992
). Data on the structural and thermodynamic consequences of Aib replacements in proteins are scarce, especially in proteinprotein 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 pepS pro interaction. Residues 313 form an
-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., 1992
). Modelling studies showed that substitution of the hydrogen by a methyl group at the C
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., 1998), improve antigen display (Sakarellos-Daitsiotis et al., 1999
) and in drug design (Hewage et al., 1999
; Howl et al., 1999
). Aib has recently been used to improve proteinprotein recognition in biologically relevant molecules such as the gp120 coat protein of HIV (Stanfield et al, 1999
) and for de novo protein design (Karle et al., 2000
).
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Materials and methods |
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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., 1996) and stored in water at 20°C. The purified S pro gave a single band on a silver-stained SDSPAGE 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 mM1 cm1 at 280 nm (Connelly et al., 1990
).
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, 1992), 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
-aminoisobutyric acid. This S16 and the A4Aib peptides were synthesized by stepwise solid-phase synthesis (Atherton and Sheppard, 1989
) 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. TFAanisoleEDT (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 wateracetonitrile 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 630°C as previously described (Connelly et al., 1990; Varadarajan et al., 1992
; Thomson et al., 1994
). Data were analyzed using the MCS-ORIGIN software package, assuming a single set of identical binding sites. Figure 1
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
X° (where X is G, H, S or Cp) =
X°mutant
X°wild-type has been followed throughout (Connelly et al., 1990
).
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Crystallization was carried out as described (Varadarajan and Richards, 1992). The final conditions in the drop were 412 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 18 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 200300 s. Table I
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, 1993
).
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Refinement was carried out using simulated annealing (SA) with the slow cooling protocol of XPLOR (Brunger, 1996) as described by Ratnaparkhi and Varadarajan (1999), with the additional calculation of Rfree (Brunger, 1992
; Kleywegt and Brunger, 1996
). Manual model building was done against
-a weighted (Read, 1986
) 2Fo Fc and Fo Fc difference Fourier maps, using FRODO (Jones, 1985
). The statistics for the final refined structures are given in Table I
. 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 B-factor plots per residue for MC and SC. Parameters such as accessibility (Connolly, 1983
) and depth (Chakravarty and Varadarajan, 1999
) were examined.
-A weighted omit maps (Read, 1986
; Brunger, 1996
) were calculated to confirm structural changes and these regions were rebuilt based on the 2Fo Fc and Fo Fc omit maps.
Quality of the structure
The final, refined structures show excellent stereochemistry (Table I) 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 I
). The quality of the structures was checked using the program PROCHECK (Laskowski et al., 1993
) 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
, values in the core and allowed regions of the Ramachandran plot.
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Results |
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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 3.335°C (Connelly et al., 1990). 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, 1986
; Connelly et al., 1990
). 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 2A
). 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, 1998
). 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, 1986
).
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Titration calorimetry has been used to characterize completely the binding thermodynamics of the S16 and the A4Aib peptides to S pro (Figure 1) under the same solution conditions as used for earlier studies (Connelly et al., 1990
).
The A4Aib peptide binding to S pro is slightly weaker at all temperatures investigated by titration calorimetry (Table II) 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 6.035.0°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 II
). 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 (50100-fold) over this temperature range. Whereas the magnitude of
G° decreases with temperature for all peptides previously studied, for A4Aib the magnitude actually increases slightly with increasing temperature. The enthalpy (
H°) of binding of A4Aib is slightly lower than that of the control peptide at all temperatures investigated (Figure 2B
). The heat capacities (
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., 1992
). At 25°C (Table III
) the thermodynamic parameters of S16 and Aib binding to S pro are similar. The
G°,
H°,
S and
Cp of binding are 0.7 kcal/mol, 2.8 kcal/mol, 6 kcal/mol.K and 60 kcal/mol.K, respectively.
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The RNase S (A4Aib) complex crystallizes in similar conditions to RNase S15 and has similar cell parameters (Table I). The electron density for Gly16 is not visible and therefore it has been deleted from the model. This is not surprising as residues 1623 are also disordered in the structure of the 20-residue S pep complexed to S protein (Kim et al., 1992
). 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, 1992
). 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., 1960
), 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 4A
).
B-factor plots also do not show significant changes (Figure 4B
). The replacement of Ala by Aib is confirmed by 2Fo Fc and Fo Fc electron density and the presence of density in omit maps (Read, 1986
; Brunger, 1996
). Figure 5A
shows the Aib side chain (ball-and-stick) in the RNase S (A4Aib) structure. Figure 5B
shows the Aib side chain positioned in the 2Fo Fc 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.
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Discussion |
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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, 1986), the binding of S pep to Spro (a model for proteinprotein 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, 1992
), 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 313 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 2A). 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
-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
S of binding is more positive for A4Aib than for S16 (Table II
). However, the
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
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
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
G°. The decrease in magnitude of
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
G° increases with temperature. The surprising lack of temperature dependence of
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 enthalpyentropy 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.
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Notes |
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3 Present address: Laboratory of Medical Biochemistry B, Department of Medical Biochemistry and Genetics, The Panum Institute, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark
4 To whom correspondence should be addressed. E-mail: varadar{at}mbu.iisc.ernet.in
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Acknowledgments |
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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.
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Received March 2, 2000; revised August 8, 2000; accepted September 14, 2000.