Interfacial Asparagine Residues within an Amide Tetrad Contribute to Max Helix-Loop-Helix Leucine Zipper Homodimer Stability
2000; Elsevier BV; Volume: 275; Issue: 48 Linguagem: Inglês
10.1074/jbc.m004264200
ISSN1083-351X
AutoresMichel Tchan, Katherine Choy, Joel P. Mackay, Alison T.L. Lyons, Naresh P.S. Bains, Anthony S. Weiss,
Tópico(s)Enzyme Structure and Function
ResumoThe transcription factor Max is the obligate dimerization partner of the Myc oncoprotein. The pivotal role of Max within the Myc regulatory network is dependent upon its ability to dimerize via the helix-loop-helix leucine zipper domain. The Max homodimer contains a tetrad of polar residues at the interface of the leucine zipper domain. A conserved interfacial Asn residue at an equivalent position in two other leucine zipper proteins has been shown to decrease homodimer stability. The unusual arrangement of this Gln-Asn/Gln′-Asn′ tetrad prompted us to investigate whether Asn92 plays a similar role in destabilizing the Max homodimer. This residue was sequentially replaced with aliphatic and charged residues. Thermal denaturation, redox time course and analytical ultracentrifugation studies show that the N92V mutation does not increase homodimer stability. Replacing this residue with negatively charged side chains in N92D and N92E destabilizes the mutant homodimer. Further replacement of Gln91 indicated that H bonding between Gln91 and Asn92 residues is not significant to the stability of the native protein. These data collectively demonstrate the central role of Asn92 in homodimer interactions. Molecular modelling studies illustrate the favorable packing of the native Asn residue at the dimer interface compared with that of the mutant Max peptides. The transcription factor Max is the obligate dimerization partner of the Myc oncoprotein. The pivotal role of Max within the Myc regulatory network is dependent upon its ability to dimerize via the helix-loop-helix leucine zipper domain. The Max homodimer contains a tetrad of polar residues at the interface of the leucine zipper domain. A conserved interfacial Asn residue at an equivalent position in two other leucine zipper proteins has been shown to decrease homodimer stability. The unusual arrangement of this Gln-Asn/Gln′-Asn′ tetrad prompted us to investigate whether Asn92 plays a similar role in destabilizing the Max homodimer. This residue was sequentially replaced with aliphatic and charged residues. Thermal denaturation, redox time course and analytical ultracentrifugation studies show that the N92V mutation does not increase homodimer stability. Replacing this residue with negatively charged side chains in N92D and N92E destabilizes the mutant homodimer. Further replacement of Gln91 indicated that H bonding between Gln91 and Asn92 residues is not significant to the stability of the native protein. These data collectively demonstrate the central role of Asn92 in homodimer interactions. Molecular modelling studies illustrate the favorable packing of the native Asn residue at the dimer interface compared with that of the mutant Max peptides. helix-loop-helix basic HLH leucine zipper glutathioneS-transferase high pressure liquid chromatography N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine electrospray mass spectrometry The human Max protein was identified by Blackwood and Eisenman (1Blackwood E.M. Eisenman R.N. Science. 1991; 251: 1211-1217Crossref PubMed Scopus (1474) Google Scholar) as the dimerization partner of the Myc oncoprotein. Heterodimerization with Max has been shown to be essential for the DNA binding, transcription activating, transformative, and apoptotic properties of Myc (2Amati B. Brooks M.W. Levy N. Littlewood T.D. Evan G.I. Land H. Cell. 1993; 72: 233-245Abstract Full Text PDF PubMed Scopus (444) Google Scholar). Max forms homodimers in addition to heterodimers with other members of the basic helix-loop-helix (HLH)1 leucine zipper (LZ) family of transcription factors. These include Mxi1 (3Zervos A.S. Gyuris J. Brent R. Cell. 1994; 72: 223-232Abstract Full Text PDF Scopus (664) Google Scholar); Mad1, Mad3, and Mad4 (4Ayer D.E. Kretzner L. Eisenman R.N. Cell. 1993; 72: 211-222Abstract Full Text PDF PubMed Scopus (623) Google Scholar, 5Hurlin P.J. Queva C. Koskinen P.J. Steingrimsson E. Ayer D.E. Copeland N.G. Jenkins N.A. Eisenman R.N. EMBO J. 1995; 14: 5646-5659Crossref PubMed Scopus (253) Google Scholar); and Mnt (6Meroni G. Reymond A. Alcalay M. Borsani G. Tanigami A. Tonlorenzi R. Nigro C.L. Messali S. Zollo M. Ledbetter D.H. Brent R. Ballabio A. Carrozzo R. EMBO J. 1997; 16: 2892-2906Crossref PubMed Scopus (127) Google Scholar, 7Hurlin P.J. Queva C. Eisenman R.N. Curr. Top. Microbiol. Immunol. 1997; 224: 115-121PubMed Google Scholar). Max has also been shown to associate with a member of the DNA-binding domain family of transcription factors, TFE-1 (8Gupta M.P. Amin C.S. Gupta M. Hay N. Zak R. Mol. Cell Biol. 1997; 17: 3924-3936Crossref PubMed Google Scholar). However, of the proteins involved in this complex regulatory network, only Max and Mnt form homodimers under physiological conditions. The role of the Max-associated bHLH LZ proteins in the regulation of cell growth, differentiation, and apoptosis depends upon dimerization with Max (9Bouchard C. Staller P. Eilers M. Trends Cell Biol. 1998; 8: 202-206Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 10Facchini L.M. Penn L.Z. FASEB J. 1998; 12: 633-651Crossref PubMed Scopus (333) Google Scholar). The HLH family of proteins is characterized by a domain consisting of two amphipathic α-helices joined by a short, nonconserved loop of random coil. HLH dimers form a globular fold of four interacting α-helices (11Wendt H. Thomas R.M. Ellenberger T. J. Biol. Chem. 1998; 273: 5735-5743Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 12Fairman R. Beran-Steed R.K. Handel T.M. Protein Sci. 1997; 6: 175-184Crossref PubMed Scopus (20) Google Scholar) in which stability and specificity are mediated through highly conserved hydrophobic residues at the core of the HLH domain (13Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (702) Google Scholar). The leucine zipper is a dimerization motif characterized by a heptad repeat of leucine residues (14Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2537) Google Scholar, 15Alber T. Curr. Opin. Gen. Dev. 1992; 2: 205-210Crossref PubMed Scopus (250) Google Scholar). Folded dimers form a parallel, coiled-coil of α-helices in which, by conventional nomenclature, (abcdefg)n, the heptad repeat Leu residues occupy position d at the dimer interface. Leu is highly conserved at this position throughout the LZ family of proteins, presumably because its side chain size and structure is such that it provides optimal hydrophobic packing. Another heptad repeat of hydrophobic residues, typically the β-branched amino acids such as Ile and Val, are found in position a at the dimer interface. There is debate over the role of interhelical salt bridges between charged residues in positions e and g in the coiled-coil. Unfavorable e-g′ electrostatic interactions, where an apostrophe indicates a residue from the apposing chain in the dimer, have been shown to inhibit dimer formation (16O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1279) Google Scholar) and potentially promote strand exchange. However, the presence of attractivee-g′ ion pairs may not contribute significantly to dimer stability; indeed, they may be less stabilizing than an equivalent neutral charge interaction (17Lumb K.J. Kim P.S. Biochemistry. 1995; 34: 8642-8648Crossref PubMed Scopus (302) Google Scholar). The HLH LZ dimer interacts as a parallel, left-handed, four-helix bundle, with each monomer consisting of two right-handed α-helices joined by the loop region of the HLH domain. The LZ extends as a coiled-coil out of the globular fold formed by the HLH domain. Crystal structures of two HLH LZ proteins, USF and Max, show that the dimerization domain is a single, contiguous functional element, with helix-2 of the HLH extending into the LZ such that there is no clear demarcation between the two domains (18Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar, 19Ferre-D'Amare A.R. Pognonec P. Roeder R.G. Burley S.K. EMBO J. 1994; 13: 180-189Crossref PubMed Scopus (332) Google Scholar, 20Brownlie P. Ceska T. Lamers M. Romier C. Stier G. Teo H. Suck D. Structure. 1997; 5: 509-520Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Horiuchi et al. (21Horiuchi M. Kurihara Y. Katahira M. Maeda T. Saito T. Uesugi S. J. Biochem. ( Tokyo ). 1997; 122: 711-716Crossref PubMed Scopus (23) Google Scholar) have shown that the Max bHLH-Z domain homodimerizes cooperatively in a manner consistent with a two-state monomer to dimer association. There is also evidence that the Max HLH LZ is able to interact as a tetramer under certain conditions (20Brownlie P. Ceska T. Lamers M. Romier C. Stier G. Teo H. Suck D. Structure. 1997; 5: 509-520Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 22Ferre-D'Amare A.R. Burley S.K. Structure. 1994; 2: 357-359Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The majority of LZ proteins contain an a position Asn residue that confers dimer specificity at the expense of stability and higher order oligomer formation. Mutation of this interfacial polar residue to a Val residue in the GCN4 LZ dramatically stabilizes the coiled-coil and promotes the formation of higher order oligomers (23Harbury P.B. Zhang T. Kim P.S. Alber T. Science. 1993; 262: 1401-1407Crossref PubMed Scopus (1338) Google Scholar). NMR studies of a recombinant Jun LZ peptide showed that its interfacial Asn residue is hydrogen bonded (H bonded) and adopts two distinct, rapidly exchanging conformations in solution (24Junius F.K. Mackay J.P. Bubb W.A. Jensen S.A. Weiss A.S. King G.F. Biochemistry. 1995; 34: 6164-6174Crossref PubMed Scopus (69) Google Scholar). Mutation of this Asn to a Leu residue caused changes in dimer stability and oligomerization status comparable with the behavior of the Asn to Val mutant of GCN4. Max contains two such a position Asn residues, Asn78 and Asn92. These residues may play a destabilizing role akin to that of the interfacial Asn in Jun and GCN4. In addition to the classical hydrophobic interactions at the dimer interface, the LZ domain of the Max homodimer exhibits unusual structural features (18Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar, 20Brownlie P. Ceska T. Lamers M. Romier C. Stier G. Teo H. Suck D. Structure. 1997; 5: 509-520Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Two regions in particular are unique among the known LZ proteins: an interfacial His-His′ packing at position 81, and a tetrad between Gln-Asn/Gln′-Asn′ at positions 91 and 92 toward the C terminus of the LZ. These residues are likely to be responsible for the propensity of Max to form a variety of heterodimers rather than Max-Max homodimers. Studies using isolated LZ domains (25Muhle-Goll C. Nilges M. Pastore A. Biochemistry. 1995; 34: 13554-13564Crossref PubMed Scopus (45) Google Scholar, 26Lavigne P. Kondejewski L.H. Houston M.E.J. Sonnichsen F.D. Lix B. Skyes B.D. Hodges R.S. Kay C.M. J. Mol. Biol. 1995; 254: 505-520Crossref PubMed Scopus (98) Google Scholar) have analyzed the role of these two regions in the context of the Myc-Max heterodimer. These indicate that a buried salt bridge between His81 of the Max LZ and two opposing glutamate residues of the Myc LZ resolves the problem of having these destabilizing hydrophilic residues at the dimer interface. The proton NMR structure of the Myc-Max LZ heterodimer (27Lavigne P. Crump M.P. Gagne S.M. Hodges R.S. Kay C.M. Sykes B.D. J. Mol. Biol. 1998; 281: 165-181Crossref PubMed Scopus (87) Google Scholar) shows Asn92 of Max forming an H bond with the backbone carbonyl of Leu420 of the Myc LZ, thus allowing the burial of the polar residue within the hydrophobic core of the LZ. Our study sheds light on the function and importance of Asn92 in the context of the Max dimerization domain. The position of this residue at the dimer interface makes it a likely candidate for one of the destabilizing residues important in facilitating peptide exchange between members of the bHLH LZ regulatory network. There is the potential for an array of H bonds to be formed between the Gln-Asn/Gln′-Asn′ tetrad of residues, in contrast to the relatively simple Asn/Asn′ interaction seen previously in the Jun and GCN4 homodimers. This unusual arrangement makes it an attractive target for mutagenesis studies, with the goal of dissecting the role of Asn92 in Max HLH LZ homodimerization. A synthetic gene was designed which encoded the HLH and LZ domains of the human Max protein (amino acids 37–105) 2The amino acid sequence of this protein corresponds to Swiss-Prot number P25912 (1Blackwood E.M. Eisenman R.N. Science. 1991; 251: 1211-1217Crossref PubMed Scopus (1474) Google Scholar). with an additional Gly-Gly-Gly at the N terminus and Gly-Gly-Cys at the C terminus.BamHI sites were incorporated onto the 5′ and 3′ ends of the coding sequence to permit insertion into the pGEX-2T expression vector (28Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). The final DNA sequence was optimized for bacterial expression by replacing rare codons with those frequently used in Escherichia coli (29Martin S.L. Vrhovski B. Weiss A.S. Gene ( Amst. ). 1995; 154: 159-166Crossref PubMed Scopus (168) Google Scholar). pGEXMax+G was isolated by expression screening, and its identity was confirmed by automated DNA sequencing in both strands. Mutagenic polymerase chain reaction (30Cadwell R.C. Joyce G.F. Genome Res. 1994; 3: S136-S140Crossref Scopus (318) Google Scholar) was used to introduce changes into pGEXMax+G, yielding the aspartate (pGEXMaxV), glutamate (pGEXMaxE), and valine (pGEXMaxV) mutant clones used in this study. Polymerase chain reaction primers were designed to remove the Gly-Gly-Gly encoding from the 5′ region of HLH LZ sequences. Polymerase chain reaction was also used to create an additional clone encoding the native Max HLH LZ sequence without the N-terminal Gly-Gly-Gly (pGEXMaxN). The four mutagenic primers were: 1) GGATCCGACCACATCAAAGACTCCTTC, 2) CTGAAACGTCAGGA(C/A)GCTCTGCTGGAA, 3) TTCCAGCAGAGCAACCTGACGTTTCAG, and 4) GGATTCTAATAGTGATCACTATTAGCAAC. Four additional mutagenic primers were used to make the two Q91A mutant forms: 5) CAGAGCGTTAGCACGTTTCAG, 6) CTGAAACGTGCTAACGCTCTG, 7) CAGAGCCACAGCACGTTTCAG, and 8) CTGAAACGTGCTGTGGCTCTG. Expression screening isolated mutant clones and automated DNA sequencing of both strands confirmed their identities. Each GST-Max fusion protein was overexpressed in E. coli DH5α that also contained pBS536. pBS536 contains the 2.2-kb GroE+EcoRI-HindIII fragment of pOF39 (MGG 202, 435–445) inserted at the EcoRV site within thetet gene of pACYC184 in an orientation that does not permit its expression from the tetpromoter. 3B. P. Surin and N. E. Dixon, unpublished results. Coexpression of GroEL and GroES from pBS536 increased the amount of soluble GST-Max proteins produced to approximately 50% of total expressed fusion protein (data not shown). Cells were induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside and grown at 37 °C for 3 h prior to harvesting. GST-Max fusion proteins were separated from cellular proteins by glutathione-agarose affinity chromatography, and Max proteins were released from the fusion protein at the engineered thrombin site with bovine thrombin. Max proteins were purified by HPLC on a semi-preparative Vydac C-18 column with a linear gradient from 25 to 45% acetonitrile 1% (v/v) trifluoroacetic acid, over 30 min. Following HPLC, the protein preparations used in this study were confirmed to be pure by Tricine gel electrophoresis and of the correct molecular weight by ESMS (Australian Government Analytical Laboratories). Each homodimer was diluted from stock solutions into physiological ionic salt buffer (50 mmTris·HCl, 125 mm NaCl, 1 mm EDTA, pH 7.4) to a final peptide concentration of 0.1 mg/ml. Stock peptide concentrations were determined by UV absorbance at 280 nm (A 280) using the extinction coefficient value of 2620 cm−1m−1 for each Max peptide. Far ultraviolet CD spectra from 200 to 260 nm were collected on a Jasco J-720 spectropolarimeter flushed continuously with N2 and routinely calibrated withd-(+)-camphor-10-sulfonic acid. For thermal denaturation profiles, ellipticity at 222 nm for each of the disulfide bridged homodimers was measured over a linear temperature gradient from 10 to 95 °C at 1 °C/min. Additional thermal denaturation data were collected for MaxV over a number of peptide concentrations: 1.0, 0.05, 0.01, and 0.005 mg/ml. Base-line spectra of physiological ionic salt buffer were collected and subtracted from all data prior to conversion into mean residue weight ellipticity values using the following formula. [θ]=θ·100·MRWc·dEquation 1 where mean residue weight ellipticity ([θ]) is expressed in deg·cm−2·dmol−1, θ is raw ellipticity values, MRW is the molecular weight divided by the number of residues in the peptide, c is the peptide concentration in mg/ml, and d is the pathlength in centimetres through the optical cell (26Lavigne P. Kondejewski L.H. Houston M.E.J. Sonnichsen F.D. Lix B. Skyes B.D. Hodges R.S. Kay C.M. J. Mol. Biol. 1995; 254: 505-520Crossref PubMed Scopus (98) Google Scholar). Estimates of α-helical content were generated by the K2D program (31Merelo J.J. Andrade M.A. Prieto A. Morán F. Neurocomputing. 1994; 6: 443-454Crossref Scopus (124) Google Scholar, 32Andrade M.A. Chacon P. Merelo J.J. Morán F. Protein Eng. 1993; 6: 383-390Crossref PubMed Scopus (949) Google Scholar). The thermal denaturation profile was fitted to either sigmoidal function (i) for MaxN and MaxE or (ii) in the case of MaxV. The thermal denaturation profile of MaxD was not subjected to model fitting.[θ] 222=a+bT−{(c+dT)−(a+bT)}exp(m(T−Tm))1+exp(m(T−Tm))Equation 2 [θ] 222=a+bT−{(c+dT)−(a+bT)}exp(m(T−Tm))1+exp(m(T−Tm))+c+dT−{(c+dT)−(c+dT)}exp(m2(T−Tm))1+exp(m2(T−Tm2))Equation 3 Analytical Ultracentrifugation—The self-association of each Max protein was investigated by sedimentation equilibrium methods on a Beckman XL-A analytical ultracentrifuge. Experiments were carried out at loading concentrations of 150, 125, 41.5, and 13.2 μm, using an An-60ti rotor spinning at 20,000, and 25,000 rpm and at a temperature of 25 °C. Samples were made up as solutions in physiological ionic salt buffer. Sedimentation equilibrium data were collected in double sector cells as absorbance versus radius scans (0.001-cm increments, 10 scans). Scans were collected at 4-h intervals and compared with determine when the samples had reached equilibrium. Analysis of the data was carried out using the NONLIN software (33Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar), and the final parameters were determined by simultaneous nonlinear least squares fits of all data sets to either a single species (disulfide-bonded dimer) or a dimer-tetramer model. The goodness of fit was determined by examination of the residuals from the fits and consideration of the χ2 values. Partial specific volumes were calculated from the amino acid sequence, and the solvent density was calculated to be 1.00482 g ml−1; both calculations were carried out using the program SEDNTERP (34Hayes D.B. Laue T. Philo J. SEDNTERP. University of New Hampshire, Durham, NH1995Google Scholar). Owing to its additional Gly residues, Max+G was used in place of MaxN for redox experiments to facilitate identification of each protein species. Approximately equimolar amounts of each disulfide-linked HLH LZ homodimer were incubated in redox buffer containing 50 mm Tris·HCl, 125 mm NaCl, 1 mm EDTA, 250 μmglutathione (oxidized), 250 μm glutathione (reduced), pH 8.3. Dissolved O2 was removed from the reaction mixture by 10 min of continuous N2 infusion. The reaction mixture was incubated at 37 °C under N2 in an airtight tube. 100-μl samples were taken at 0, 3, and 16 h, and a 300-μl sample was taken after 4 days. Samples were analyzed by HPLC on an analytical Delta Pak C-18 column (Waters, Milford, MA) with a linear gradient identical to that used for peptide purification. Peaks were collected and lyophilized, and their identities were confirmed by ESMS. The calculated molecular weights for each species are as follows: Max+G homodimer (17,293), Max+G-glutathione conjugate (8,953), MaxD homodimer (16,952), MaxD-glutathione conjugate (8,783), MaxE homodimer (16,980), MaxE-glutathione conjugate (8,797), MaxV homodimer (16,918), and MaxV-glutathione conjugate (8,766). Molecular models were created on a Silicon Graphics workstation using the InsightII suite of programs (version 97.0, Molecular Simulations Inc., 1997). The crystal structure of the HLH LZ domains of the Max homodimer bound to its cognate DNA fragment (18Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar) was used as a template. Hydrogen atoms were added to the crystal structure at standard chemical positions. Two residues were replaced; these were Ala residues present in the pdb coordinates but not present in the originally published human Max sequence (1Blackwood E.M. Eisenman R.N. Science. 1991; 251: 1211-1217Crossref PubMed Scopus (1474) Google Scholar). The two Ala residues in each monomer were replaced by His79 and Gln82. Asn92 was replaced by either Asp, Glu, or Val residues to create the three mutant model homodimers-MaxD, MaxE, and MaxV. The bound DNA fragment was removed from the system prior to energy minimization calculations. Each model was immersed in a 40 × 115 × 40 Å box filled with water molecules. The potential energy of the molecular system was minimized using the consistent valence force field (35Dauber-Osguthorpe P. Roberts V.A. Osguthorpe D.J. Wolff J. Genest M. Hagler A.T. Proteins. 1988; 4: 31-47Crossref PubMed Scopus (1937) Google Scholar). Energy minimization of the homodimer models was performed as a stepwise process designed to gradually release the molecule from restraints tethering it to Max crystal structure coordinates (36Biosym Technologies InsightII User Guide , Version 2.3.0. Biosym Technologies, San Diego, CA1993Google Scholar). Hydrogen atoms were relaxed using Steepest Descents for 20 iterations; all other atoms were tethered to their original positions. Peptide side chain atoms and oxygen atoms of water molecules were then released from tethering restraints and energy minimized for a further 20 iterations using the same algorithm. Restraints were released from the rest of the molecule for the final energy minimization of 300 iterations using conjugate gradients. The stereochemical quality of each model was assessed using the program Procheck (37Laskowski R.A. Moss D.S. Thornton J.M. J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1083) Google Scholar). The CD spectra at 10 °C for each of the peptides is shown in Fig. 1 A. α-Helicity estimates were 91% for the native protein (MaxN); and 20, 21, and 76% for the mutant proteins, N92→D (MaxD), N92→E (MaxE), and N92→V (MaxV), respectively. Thermal denaturation of the disulfide bridged homodimers was followed by measurements of [θ]222 from 10 to 95 °C (Fig.1 B). Decreases in absolute [θ]222 correlate with decreasing α-helicity and the formation of monomeric peptides from the initial homodimers. Each protein underwent a decrease in helicity and a shift to an increasingly random structure as it was heated. However, the initial helicities and unfolding pathways differed for each of the peptides studied. MaxN shows a cooperative unfolding event with a melting temperature (T m) of 57 °C. T m is defined as the temperature at which half the peptide population is folded. The thermal denaturation profile exhibited good correlation to the theoretical curve. MaxE also displayed a thermal denaturation profile in keeping with a cooperative mechanism of unfolding. However, the exchange of the polar Asn residue for the more bulky, charged side chain of Glu had a strong destabilizing effect. At 10 °C, MaxE showed less ellipticity than MaxN, indicating that the population of MaxE molecules contained less helical secondary structure. The T m of MaxE was 37 °C, considerably lower than that of MaxN. MaxV exhibited an unfolding pattern similar to that of MaxN up to approximately 85 °C (Fig. 1 B). From 85 to 95 °C, MaxV showed the beginning of a second unfolding transition (Fig.1 C). This second transition was reproducible from peptide concentrations of 0.005–1.0 mg/ml and throughout the series of 22 separate thermal denaturation experiments performed on MaxV. Although the helicity of MaxV at 10 °C was not as marked as that of the native peptide, the Val mutant undergoes its initial denaturation event at approximately the same temperature, 58 °C compared with 57 °C for MaxN. The second transition had an approximateT m of 90 °C, although this is only an estimate because the ellipticity values had not plateaued by 95 °C, which is the upper limit of the CD spectrometer. Additional thermal denaturation experiments performed with MaxV in physiological ionic salt plus 4 m urea failed to clarify the characteristics of this second unfolding event (data not shown). The second transition displayed by MaxV contrasts significantly with the profiles exhibited by the other peptides. For this reason, we wished to exclude the possibility of higher order interactions. TheT m for the first denaturation transition of MaxV was concentration independent across the range of samples studied (Fig.2 A). Data from sedimentation equilibrium measurements carried out on MaxV fitted well to a single species model where the only species present was the disulfide-bonded MaxV dimer with an apparent molecular weight of 18300 ± 1700 Da (Fig. 2 B). Fitting the data to a monomer-dimer equilibrium (disulfide bridged dimer-tetramer) gave a marginally better fit, with a dimerization constant of 420 m−1. However, under these conditions the protein would be >99% dimeric at 0.1 mg/ml. We propose that both models fitted the data similarly because although the propensity of MaxV to form higher order aggregates appears to be real, under our experimental conditions it is small enough that a single species dimeric model describes the interaction adequately. Taken together, these results indicate that MaxV is present predominantly as a disulfide bridged dimer under the conditions of this study. MaxD was predominantly unfolded for the duration of the thermal denaturation experiment (Fig. 1 B). The unfolding pathway suggests multiple points of slight inflection, indicating that the small amount of helical MaxD present melts in a sequential manner consistent with subdomains unfolding within the HLH LZ. The melting temperatures of these inflection points were not determined because it is apparent that MaxD does not display a dimer to monomer unfolding pattern under these conditions. To exclude the possibility that the C-terminal disulfide bond was artificially imparting the correct dimer alignment and thus masking any role of the Asn residue in orientating the molecules, the thermal denaturation profiles of MaxN and MaxV were examined under the reducing conditions imparted by a 5-fold excess of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Neither denaturation profile varied significantly from those of their respective disulfide bridged homodimers (data not shown). Two additional mutant proteins were made to assess whether Gln91 influenced the stability of the native or Val mutant proteins. These were MaxAN and MaxAV, in which Gln91 was replaced by Ala in MaxN and MaxV, respectively. Thermal denaturation of MaxAN and MaxAV was followed by measurements of [θ]222from 10 to 95 °C (Fig. 3). Both molecules showed unfolding profiles similar to those of MaxN and MaxV. MaxAN had a T m of 57 °C, which compared closely to that of MaxN (58 °C). MaxAV similarly unfolded at a comparable temperature to that of MaxV (57 and 58 °C, respectively). To determine whether there was preferential formation of homodimer or heterodimer species between mutant Max forms and the native Max sequence, dimerization behavior was studied under redox conditions. By virtue of additional Gly residues at the N terminus of the native sequence, Max+G was used in place of MaxN for redox experiments. This facilitated identification of each protein species. In each case, the native peptide showed a weak propensity to form mixed disulfides with glutathione from an initially homodimeric state. After 4 days, 5–14% of Max+G was covalently bound to glutathione. MaxD showed a strong preference to dissociate from an initially homodimeric form into a mixed disulfide with glutathione (MaxD-SG). The MaxD-SG form was evident after only 3 h. After 4 days, 46% of the MaxD peptide was present as a glutathione-bound monomer (Fig.4). Max+G also formed mixed disulfides with glutathione, however, to a lesser degree than MaxD; after 4 days, only 5% of the Max+G peptide was present in this form. Max+G showed no propensity to interact with MaxD. MaxE behaved similarly to MaxD. It showed the presence of a small amount of glutathione adduct after 3 h, and after 4 days this had increased to 68% of the MaxE peptide (Fig.5). Max+G again displayed a weak propensity to dissociate from its homodimeric form and react with glutathione. After 4 days, 14% of the Max+G peptide was covalently bound to glutathione. ESMS analysis of the minor peak 4 identified two contributing species, suggesting a mixture of dimer components. The MaxV mutant displayed a similar monomer-dimer distribution to that of the native Max+G protein. After 4 days, 9% of the Max+G peptide and 5% o
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