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The Role of the Hinge Loop in Domain Swapping

2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês

10.1074/jbc.m413157200

1083-351X

Delia Picone, Anna Di Fiore, Carmine Ercole, M. Franzese, Filomena Sica, Simona Tomaselli, L. Mazzarella,

Bacterial Genetics and Biotechnology

Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16–22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase. Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16–22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase. Domain swapping, a process by which two or more protein molecules exchange identical structural elements to form dimers or higher oligomers (1.Schlunegger M.P. Bennet M.J. Eisenberg D. Adv. Prot. Chem. 1997; 50: 61-122Crossref PubMed Google Scholar), has been observed in an increasing number of proteins. More than 50 crystal structures of domain-swapped proteins have been deposited in PDB 1The abbreviations used are: PDB, Protein Data Bank; BS-RNase, bovine seminal ribonuclease; CD, circular dichroism spectroscopy; mBS, monomeric N67D variant of BS-RNase with cysteines 31 and 32 linked to glutathione moieties; RMSD, root-mean-square deviation; RNase A, bovine pancreatic ribonuclease; Ala19-mBS, P19A variant of mBS; Ser16-Thr17-Ala19-Ala20-mBS, G16S/N17T/P19A/S20A variant of mBS; Ala19-BS-RNase, P19A variant of BS-RNase; Ser16-Thr17-Ala19-Ala20-BS-RNase, G16S/N17T/P19A/S20A variant of BS-RNase; TOCSY, total correlation spectroscopy; PEG, polyethylene glycol. since the first x-ray structure of a protein showing the interchange of N-terminal regions between the two polypeptide chains was reported (2.Mazzarella L. Capasso S. Demasi D. Di Lorenzo G. Mattia C.A. Zagari A. Acta Crystallogr. Sect. D. 1993; 49: 389-402Crossref PubMed Google Scholar). However, the possible physiological significance of this phenomenon is still unclear (3.Rousseau F. Schymkovitz J.W.H. Itzaki L.S. Structure. 2003; 11: 243-251Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). It has been proposed that a large number of proteins may undergo this process under physiological or pathological conditions; thus it could represent a mechanism to regulate function, or even an evolutionary strategy to increase protein complexity. In the panorama of the swapping proteins a special case is represented by bovine seminal ribonuclease (BS-RNase), a homodimeric protein in which the two subunits are covalently linked through two disulfide bridges between cysteines 31 and 32 of one subunit with cysteines 32 and 31 of the partner subunit, respectively (4.D'Alessio G. Di Donato A. Mazzarella L. Piccoli R. D'Alessio G. Riordan J.F. Ribonucleases: Structures and Functions. Academic Press, New York.1997: 383-423Crossref Google Scholar). In this protein the swapping process involves two dimers, in which the two subunits change their tertiary structure within a basically invariant quaternary assembly imposed by the two interchain disulfides: in the dimer-dubbed MxM the N-terminal arms (residues 1–15) are exchanged, or swapped, between the two subunits, whereas in the dimer indicated as M=M no swapping occurs. Thus, in this particular system the swapping phenomenon does not depend on the overall concentration of the protein. Furthermore, in the quaternary structure of MxM, the acceptable values of the end-to-end distance, spanned by the hinge peptide, are almost as sharply restricted as they are within the tertiary structure of the unswapped dimer. This finding is at variance with what is usually observed in the swapping process, where a monomer to dimer (M/D) transition is commonly observed and the swapped dimer often presents a considerable degree of flexibility and, therefore, a certain degree of variability of the end-to-end distance of the hinge peptide. In the latter case, the swapped state is expected to become statistically more favored as the rigidity of the hinge is increased. Indeed, this argument has been used to explain the elevated frequency of proline in the hinge peptide sequence of proteins prone to swap (5.Bergdoll M. Remy M.H. Cagnon C. Masson J.M. Dumas P. Structure. 1997; 5: 391-401Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Furthermore, in an M/D transformation other parameters may influence significantly the process, such as the nature and the extent of "O-interface," i.e. the additional interface formed in the dimer and exposed to the solvent in the monomer (1.Schlunegger M.P. Bennet M.J. Eisenberg D. Adv. Prot. Chem. 1997; 50: 61-122Crossref PubMed Google Scholar). In the MxM/M=M equilibrium of BS-RNase, the quaternary structure is highly preserved and the O-interface 2Here an extensive use is made of this term, with reference to a monomeric species prior to its conversion to the unswapped (M=M) or to the swapped (MxM) species. varies only for the different conformation adopted by the hinge peptide in the two dimers (6.Berisio R. Sica F. De Lorenzo C. Di Fiore A. Piccoli R. Zagari A. Mazzarella L. FEBS Lett. 2003; 554: 105-110Crossref PubMed Scopus (26) Google Scholar). This feature offers the unique opportunity to isolate the effects of the hinge sequence on the equilibrium ratio between MxM and M=M. In the native protein the relative amount of the two dimers is about 70:30 (7.Piccoli R. Tamburrini M. Piccialli G. Di Donato A. Parente A. D'Alessio G. Proc. Natl. Acad. Sci. 1992; 89: 1870-1874Crossref PubMed Scopus (135) Google Scholar). Interestingly, the selective reduction of the interchain disulfides produces the species which could be involved into a M/D swapping transformation, as MxM gives rise to a non-covalent swapped dimer (NCD), whereas M=M readily dissociates into monomers. BS-RNase sequence is 81% identical to that of bovine pancreatic ribonuclease (RNase A), the first protein that was shown to dimerize via the N termini interchange between the two intervening chains, upon lyophilization in acetic acid (8.Crestfield A.M. Stein W.H. Moore S. Arch. Biochem. Biophys. 1962; 1: 217-222PubMed Google Scholar). For this protein, however, more recent experiments have demonstrated that only a minor fraction of the dimers is swapped at the N terminus, whereas a major fraction is swapped at the C terminus (9.Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Crossref PubMed Scopus (75) Google Scholar, 10.Liu Y. Hart P.J. Schlunegger M.P. Eisenberg D. Proc. Natl. Acad. Sci. 1998; 95: 3437-3442Crossref PubMed Scopus (184) Google Scholar, 11.Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Crossref PubMed Scopus (267) Google Scholar). In the former dimer the contacts at the so-called "C-interface," i.e. the interface between the swapped domain (residues 1–15) and the major domain (residues 23–124), are identical to those found in the covalent dimers of BS-RNase. The sequence alignment of the two proteins shows that four substitutions out of a total of 23 are located in the 16–22-hinge region. In order to clarify the actual role of the hinge sequence in the swapping process of BS-RNase, a homologue-scanning mutagenesis approach has been followed, using as reference the sequence of RNase A. We have prepared two mutants, Ala19-BS-RNase and Ser16-Thr17-Ala19-Ala20-BS-RNase, in which either Pro19 or all four residues of the BS-RNase sequence have been substituted with the corresponding ones of the pancreatic enzyme. Here we report the x-ray structures of the MxM form of the two mutants and discuss the results on the basis of the MxM/M=M equilibrium data measured in solution for Ser16-Thr17-Ala19-Ala20-BS-RNase and those previously published for Ala19-BS-RNase and for the parent BS-RNase (12.Ercole C. Avitabile F. del Vecchio P. Crescenzi O. Tancredi T. Picone D. Eur. J. Biochem. 2003; 270: 4729-4735Crossref PubMed Scopus (19) Google Scholar). Site-directed Mutagenesis—Site-directed mutagenesis was performed using a megaprimer polymerase chain reaction method (13.Ke S. Madison E. Nucleic Acids Res. 1997; 25: 3371-3372Crossref PubMed Scopus (228) Google Scholar) to produce the mutants coding for G16S/N17T/P19A BS-RNase and G16S/N17T/P19A/S20A BS-RNase, starting from the pET-22b(+) plasmid cDNA coding for the P19A BS-RNase (12.Ercole C. Avitabile F. del Vecchio P. Crescenzi O. Tancredi T. Picone D. Eur. J. Biochem. 2003; 270: 4729-4735Crossref PubMed Scopus (19) Google Scholar). PCR amplification was performed with an Eppendorf Mastercycler amplifier as previously described (12.Ercole C. Avitabile F. del Vecchio P. Crescenzi O. Tancredi T. Picone D. Eur. J. Biochem. 2003; 270: 4729-4735Crossref PubMed Scopus (19) Google Scholar). For the tetramutant the two mutagenic primers 5′-TAGCAGAGGTGCTGCTGTC-3′ and 5′-AAGAGCTACCAGCAGAG-3′ were used in succession (nucleotides that represent mutations are underlined). The amplified, mutated genes were separated, excised, and purified from the agarose gel followed by cloning into the pET-22b(+) plasmid between HindIII and NdeI sites. Mutations were confirmed by DNA sequencing. To avoid heterogeneity (see "Results and Discussion"), the basic sequence of BS-RNase contains the substitution of Asn67 with an aspartic residue. This modified sequence, together with the N-terminal Met, constitutes the parent protein (henceforth referred to as mBS for the monomeric species or BS-RNase for the dimers), whereas the unmodified one is referred to as native protein. Recovery of Proteins—The proteins were expressed in Escherichia coli and purified in monomeric form, with cysteines 31 and 32 linked to two glutathione molecules, as previously described (14.Crescenzi O. Carotenuto A. D'Ursi A.M. Tancredi T. D'Alessio G. Avitabile F. Picone D. J. Biomol. NMR. 2001; 20: 289-290Crossref PubMed Scopus (5) Google Scholar). Monomers with cysteines 31 and 32 in the reduced form were obtained by selective reduction of the mixed disulfide bridges with a 5:1 molar excess of dithiothreitol for 20 min at room temperature in 0.1 m Tris acetate buffer, pH 8.4. The samples were either carboxyamidomethylated with iodoacetamide (15.D'Alessio G. Malorni M.C. Parente A. Biochemistry. 1975; 14: 1116-1122Crossref PubMed Scopus (77) Google Scholar), to obtain the monomeric proteins used for CD, or dialyzed against 0.1 m Tris acetate, pH 8.4 for 20 h at 4 °C, to obtain dimers. The last step of the purification procedure was always a gel filtration on Sephadex G-75 to separate monomers from dimers. All dimerization steps were performed at 4 °C. Protein concentration was measured by UV spectrophotometry assuming ϵ (0.1%, 278 nm, 1 cm) = 0.5. Protein homogeneity was checked by SDS-PAGE and MALDI-TOF mass spectra, registered at "Sezione di Spettrometria di Massa" of the CIMCF, University of Naples Federico II. The correct folding of all the monomers was checked by CD. The enzymatic activity on yeast (16.Kunitz M. J. Biol. Chem. 1946; 164: 563-568Abstract Full Text PDF PubMed Google Scholar) was comparable to that of native mBS, so confirming the correct folding of the active site. CD Spectra Measurements—CD spectra were recorded with a Jasco J-715 spectropolarimeter equipped with a Peltier type temperature control system (Model PTC-348WI). Molar ellipticity per mean residue, [θ]in deg cm2 dmol–1, was calculated from the equation: [θ] = [θ]obs·mrw/10·l·C, where [θ]obs is the ellipticity measured in degrees, mrw is the mean residue molecular mass, 117 Da (15.D'Alessio G. Malorni M.C. Parente A. Biochemistry. 1975; 14: 1116-1122Crossref PubMed Scopus (77) Google Scholar), C is the protein concentration in g l–1 and l is the optical path length of the cell in cm. A 0.1-cm path length cell and a protein concentration of about 0.3 mg ml–1 in 10 mm sodium acetate buffer, pH 5.0, were used. CD spectra were recorded at 25 °C with a time constant of 16 s, a 2-nm bandwidth, and a scan rate of 5 nm min–1; they were signal-averaged over five scans at least, and baseline corrected by subtracting the buffer spectrum. Thermal unfolding curves were recorded in the temperature scan mode at 222 nm from 25 up to 85 °C with a scan rate of 1.0 K min–1. NMR—NMR measurements were performed on a Bruker DRX500 spectrometer. All spectra were collected using the standard Bruker pulse sequence library. Protein concentration was 2 mm in 95% H2O, 5% D2O, pH 5.65. Spectra were processed with NMRPipe (17.Delaglio F. Grzesiek S. Vuister G. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11630) Google Scholar) and analyzed with NMRView (18.Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2686) Google Scholar) programs. Kinetics of Interconversion of Dimeric Forms—To follow the interconversion kinetics, dimer samples were incubated at 37 °C. At given times, aliquots were withdrawn, the interchain disulfide bridges were selectively reduced as described (7.Piccoli R. Tamburrini M. Piccialli G. Di Donato A. Parente A. D'Alessio G. Proc. Natl. Acad. Sci. 1992; 89: 1870-1874Crossref PubMed Scopus (135) Google Scholar), and the mixture was chromatographed on an analytical Superdex 75 HR 10/30 column (Amersham Biosciences). The amount of MxM and M=M was evaluated by integrating the peaks of dimer and monomer, respectively. Extent of the N-terminal Swapping at Equilibrium—Cross-linking experiments were done using divinyl sulfone (DVS) as a 10% solution in ethanol. The dimers (20 μg) in sodium acetate buffer (100 mm, pH5, 100 μl) and DVS (1 μl of the 10% solution) were incubated at 30 °C (19.Ciglic M.I. Jackson P.J. Raillard S.I. Haugg M. Jermann T.M. Opitz J.G. Trabesinger-Ruf N. Benner S.A. Biochemistry. 1998; 37: 4008-4022Crossref PubMed Google Scholar). This is approximately a 1,000-fold excess of sulfone to each subunit of the protein. Aliquots were withdrawn over a period of 96 h, quenched with 2-mercaptoethanol (final concentration 200 mm), incubated for 15–30 min at room temperature, and loaded on a gel for reducing SDS-PAGE. A qualitative estimation of the monomer to cross-linked dimer ratio was obtained by Coomassie Blue staining. Crystallization and Data Collection—The swapped dimers of Ala19-BS-RNase and Ser16-Thr17-Ala19-Ala20-BS-RNase variants were crystallized using protein solutions containing the equilibrium mixture of the swapped and unswapped form without further purification. In detail, Ala19-BS-RNase and Ser16-Thr17-Ala19-Ala20-BS-RNase crystals were grown at room temperature by the vapor diffusion sitting drop method. Equal volumes of protein (27 mg/ml) and of a solution containing 30% (w/v) PEG 4000, 0.1 m Tris-HCl, pH 8.5 and 0.2 m sodium acetate were mixed and equilibrated against a 750-μl reservoir containing the same precipitant solution. Single crystals of the two mutants were obtained after 1 day and present very similar morphology. They belong to the orthorhombic space group P212121 and are isomorphous to the wild-type protein crystallized under very similar conditions (PDB code 1R5D) (20.Merlino A. Vitagliano L. Sica F. Zagari A. Mazzarella L. Biopolymers. 2004; 73: 689-695Crossref PubMed Scopus (20) Google Scholar). Diffraction data were collected on a ENRAF-NONIUS DIP area detector equipped with a FR591 rotating anode of the Istituto di Biostrutture e Bioimmagini (CNR, Naples). Ala19-BS-RNase data collection was carried out at room temperature up to 2.20-Å resolution. Because of the lower resolution of the diffraction data, a Ser16-Thr17-Ala19-Ala20-BS-RNase crystal was cryo-cooled at 100 K by using 18% (v/v) glycerol as cryo-protector, and the diffraction data were collected up to the same resolution (2.20 Å). The two data sets were processed and scaled with the HKL package (21.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar). Crystal parameters and data collection statistics are given in Table II.Table IIData collection and refinement statisticsAla19-BS-RNaseSer16Thr17Ala19Ala20-BS-RNaseCrystal data Space groupP212121P212121Cell parameters a (Å)49.5048.96 b (Å)62.4361.10 c (Å)81.6081.22Data collection Resolution limits (Å)20.00-2.2020.00-2.20 Highest resolution shell (Å)2.24-2.202.25-2.20 No. of observations58,42168,829 No. of unique reflections12,49212,548 Completeness (%)93.4 (93.5)aNumbers in parentheses indicate values for the highest resolution shell.98.2 (99.5) I/σ(I)18 (5)25 (7) Average multiplicity55 Rmerge (%)7.2 (24.7)5.7 (24.6) Mosaicity0.180.32Refinement results Molecules for asymmetric unit11 Resolution limits (Å)20.00-2.2020.00-2.20 Number of reflections with F>2σ(F)9,45611,641 No. of reflections in working set11,16710,439 No. of reflections in test set1,1501,202 Rworking (%)17.120.8 Rfree (%)20.225.9 No. of protein atoms1,8531,878 No. of water molecules96102RMSD from ideal values Bond lengths (Å)0.0060.008 Bond angles (°)1.241.57 Dihedral angles (°)24.8025.82 Improper angles (°)0.750.91Average B-factors (Å2) Protein, overall30.3328.66 Main chain28.8028.24 Side chain31.2528.81 Solvent atoms37.4531.48Ramachandran plot statistics Most favored regions (%)90.085.0 Additional allowed regions (%)10.014.6 Generously allowed regions (%)0.00.0 Disallowed regions (%)0.00.4a Numbers in parentheses indicate values for the highest resolution shell. Open table in a new tab Structure Determination and Refinement—The crystal structures were solved by the molecular replacement method. The atomic coordinates of the native enzyme, stripped of all solvent molecules and hinge peptide residues, were used as starting model. Structure refinement, performed by using the CNS program (22.Brunger A.T. Department of Molecular Biophysics and Biochemistry. Yale University Press, New Haven, CT1996Google Scholar), began with a cycle of rigid body refinement. In this step the starting model was divided in four groups formed by the two N-terminal helical segments (residues 1–15) and the two main bodies (residues 24–124). Each mutant was then refined by alternating positional and temperature refinement with manual building by using O (23.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The missing hinge peptide residues were built by fitting the electron density from difference Fourier maps. Water molecules were then added to the models through the automatic protocol of CNS together with the contribution of the disordered solvent. Reflections with intensity greater than 2σ(I) in all resolution range (20.00–2.20 Å for both mutants) were included in the minimization procedure. The final models of the two proteins have Rfactor/Rfree indexes of 0.171/0.202 and 0.208/0.259, respectively. The models have good geometry as evaluated with WHATCHECK (24.Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1816) Google Scholar). The full lists of refinement statistics are reported in Table II. Superimposition of the mutants with the wild-type protein was achieved by using the Cα carbon atoms of residues 2–15 and 23–124 of the two chains. Because of the swapping, the quaternary structure was compared by first superimposing one structural unit (residues 2–15 of one chain and 23–124 of the second chain) and, successively, computing the angle needed to superimpose the second structural unit. Characterization of the Monomers—All the plasmids were coding for Asp at position 67 in order to avoid heterogeneity arising from the spontaneous deamidation of Asn67, which characterizes the native enzyme (25.Di Donato A. D'Alessio G. Biochemistry. 1981; 20: 7232-7237Crossref PubMed Scopus (37) Google Scholar). Asn67 is located in a disulfide-linked octapeptide loop (65–72) exposed to the solvent and on the most far site with respect to the swapping domain and to the O-interface. This substitution in RNase A has been shown not to influence the correct folding of the chain and its thermal stability (26.Catanzano F. Graziano G. Capasso S. Barone G. Protein Sci. 1997; 6: 1682-1693Crossref PubMed Scopus (52) Google Scholar). mBS shows a similar behavior, and, in addition, the extent of the swapping phenomenon in the dimeric species is not altered (12.Ercole C. Avitabile F. del Vecchio P. Crescenzi O. Tancredi T. Picone D. Eur. J. Biochem. 2003; 270: 4729-4735Crossref PubMed Scopus (19) Google Scholar). The first products of our purification procedure were about 15 mg/liter of recombinant BS-RNase or its variants in monomeric form, with cysteines 31 and 32 linked to two glutathione molecules. The thermal stability of the two mutant proteins was monitored by CD spectroscopy. Fig. 1 reports the folded fraction (fF), calculated as (θmeasured – θunfolded)/(θfolded – θunfolded) at 222 nm, as a function of the temperature for mBS, Ala19-mBS, and Ser16-Thr17-Ala19-Ala20-mBS in comparison with that of recombinant RNase A. The values of Tm, calculated by linear regression analysis of the experimental data, indicate that both mutants display only a very small difference with respect to the parent form (Tm = 53 °C). On the other hand, the Tm of Ala19-mBS (55 °C) is 6 °C lower than that of RNase A; the difference is even higher in the mutant containing four substitutions in the hinge region (Tm = 53.5 °C), despite the greater similarity of its sequence to that of RNase A. In both cases the effect is significantly greater than that produced by the sole substitution of Asn67 (27.Catanzano F. Graziano G. Cafaro V. D'Alessio G. Di Donato A. Barone G. Biochemistry. 1997; 36: 14403-14408Crossref PubMed Scopus (23) Google Scholar). For a more accurate evaluation of the effect of the mutations on the solution structure of the monomeric proteins we resorted to two-dimensional NMR spectra. The overlay of TOCSY spectra of Ser16-Thr17-Ala19-Ala20-mBS and mBS indicated that most resonances were coincident, thus confirming that the two monomers have a very similar conformation. In the expanded regions reported in Fig. 2 it is evident the presence of three new NH-CH3 connectivities in the spectrum of Ser16-Thr17-Ala19-Ala20-mBS (black), tentatively assigned to Thr17, Ala19, and Ala20. The analysis of sequential contacts in the NOESY spectrum allowed the proton assignment of all the spin systems of the hinge residues, which is reported in Table I. The table also reports the proton resonances of the 16–22 region for mBS and for RNase A (28.Avitabile F. Alfano C. Spadaccini R. Crescenzi O. D'Ursi A.M. D'Alessio G. Tancredi T. Picone D. Biochemistry. 2003; 42: 8704-8711Crossref PubMed Scopus (23) Google Scholar) for comparison. Despite the sequence identity, the chemical shift values of Ser16-Thr17-Ala19-Ala20-mBS show a striking difference with those of RNase A, suggesting that the hinge loop has a different local environment in the two proteins.Table IProton chemical shift of the 15–21 loop residues for mBS, Ser16-Thr17-Ala19-Ala20-mBS and RNase A at pH 5.65, 300 KNo. of residuemBSSer16Thr17Ala19Ala20-mBSRNase A15SerNH8.90SerNH8.94SerNH8.89SerHA4.76SerHA4.24SerHA4.31SerHB13.79SerHB13.87SerHB13.96SerHB23.65SerHB23.74SerHB23.74GlyNH8.59SerNH8.18SerNH8.0216GlyHA14.09SerHA4.27SerHA4.32GlyHA23.87SerHB13.76SerHB1NDaND, not determined.SerHB23.69SerHB2NDAsnNH7.98ThrNH8.13ThrNH7.4617AsnHA4.63ThrHA4.20bOverlapped.ThrHA4.46AsnHB12.64ThrHB4.20bOverlapped.ThrHB4.07AsnHB22.58ThrMG1.04ThrMG0.96SerNH8.29SerNH8.75SerNH8.4118SerHA4.17SerHA4.38SerHA4.27SerHB13.63SerHB14.05SerHB1NDSerHB23.58SerHB24.00SerHB2NDProHA4.18AlaNH8.06AlaNH7.5619AlaHA4.02AlaHA3.44AlaMB1.14AlaMB1.00SerNH8.28AlaNH7.75AlaNHND20SerHA4.32AlaHA3.88AlaHANDSerHB13.78AlaMB1.00AlaMBNDSerHB23.62SerNH8.41SerNH8.30SerNH8.0421SerHA4.36SerHA4.47SerHA4.31SerHB13.90SerHB13.91SerHB13.92SerHB2NDSerHB23.78SerHB23.88a ND, not determined.b Overlapped. Open table in a new tab Although the hinge peptide was found to be flexible in RNase A too (28.Avitabile F. Alfano C. Spadaccini R. Crescenzi O. D'Ursi A.M. D'Alessio G. Tancredi T. Picone D. Biochemistry. 2003; 42: 8704-8711Crossref PubMed Scopus (23) Google Scholar), its greatly enhanced mobility in Ser16-Thr17-Ala19-Ala20-mBS clearly indicates that more substitutions external to the hinge region, such as Ser80 replaced by Arg in BS-RNase, play a role in fixing this peptide in RNase A. In conclusion, the disorder, observed for the hinge region of the monomeric derivative of BS-RNase in the solid state (29.Sica F. Di Fiore A. Zagari A. Mazzarella L. Proteins. 2003; 52: 263-271Crossref PubMed Scopus (18) Google Scholar) and in solution (28.Avitabile F. Alfano C. Spadaccini R. Crescenzi O. D'Ursi A.M. D'Alessio G. Tancredi T. Picone D. Biochemistry. 2003; 42: 8704-8711Crossref PubMed Scopus (23) Google Scholar), appears to be also a feature of the mutants. Moreover, it seems reasonable to assume that similar disorder characterizes the corresponding unswapped dimers, where each subunit shares the global fold of the monomeric derivatives. Extent of the Swapping—BS-RNase and its variants in their monomeric form, with cysteines 31 and 32 linked to two glutathione molecules, can be easily converted into dimers by selective reduction of the disulfide bridges followed by air oxidation of the exposed sulfhydryls. The characterization of the swapping process for Ala19-BS-RNase has been already reported (12.Ercole C. Avitabile F. del Vecchio P. Crescenzi O. Tancredi T. Picone D. Eur. J. Biochem. 2003; 270: 4729-4735Crossref PubMed Scopus (19) Google Scholar). Two independent methods have been used to evaluate the swapping tendency of the other mutant. The first protocol is based on the higher reactivity of the interchain disulfide bridges with respect to the intrachain ones (7.Piccoli R. Tamburrini M. Piccialli G. Di Donato A. Parente A. D'Alessio G. Proc. Natl. Acad. Sci. 1992; 89: 1870-1874Crossref PubMed Scopus (135) Google Scholar). As in the case of the parent protein and of Ala19-BS-RNase, Ser16-Thr17-Ala19-Ala20-BS-RNase folds mainly in the unswapped form, and in freshly prepared samples the MxM content is about 15%. Fig. 3 displays the course of the M=M to MxM conversion as a function of the incubation time at 37 °C. The process in Ser16-Thr17-Ala19-Ala20-BS-RNase is very similar to that reported for BS-RNase, as it reaches the same 70:30 equilibrium between swapped and unswapped dimers, except for a slight increase in the conversion rate. Fig. 3 also reports the time course of the reverse process, namely the conversion of MxM into M=M, which confirms the equilibrium ratio previously found. Both reactions were essentially complete within 2 days, whereas at 4 °C the interconversion was effectively blocked (data not shown). The extent of swapping on the equilibrium mixtures was also assessed by cross-linking experiments with divinyl sulfone (DVS), followed by SDS-PAGE analysis under reducing conditions. DVS joins covalently the two histidines of the active site (His12 and His119), which belong to the sa