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• W2036931844 abstract "Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25 °C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25 °C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations. Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25 °C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25 °C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations. Tooth enamel is one of the hardest and most heavily mineralized vertebrate tissues that can withstand wear without catastrophic failure during the entire life span of an organism (1Robinson C. Connell S. Kirkham J. Shore R. Smith A. J. Mater. Chem. 2004; 14: 2242-2248Crossref Scopus (93) Google Scholar, 2Chai H. Lee J.J. Constantino P.J. Lucas P.W. Lawn B.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7289-7293Crossref PubMed Scopus (184) Google Scholar). The formation of enamel takes place in an extracellular environment, including an array of complex proteins and proteases in three main stages, namely the secretory, transition, and maturation stages (3Simmer J.P. Fincham A.G. Crit. Rev. Oral Biol. Med. 1995; 6: 84-108Crossref PubMed Scopus (353) Google Scholar). As the enamel development progresses, proteases such as enamelysin (MMP-20) and later kallikrein 4 (KLK-4) cleave the proteins, which are removed from the mineralization site in the extracellular matrix enabling the growth of enamel crystals and resulting in enamel hardness. Thus, during amelogenesis, the soft mineralized tissue formed during the early stages becomes hard and tough and almost devoid of any proteins at the maturation stage. Amelogenin is the major constituent (∼90%) of the protein matrix during the secretory stage and, together with other proteins in enamel, is responsible for the hierarchical structure observed in the enamel prisms (4Snead M.L. Zeichner-David M. Chandra T. Robson K.J. Woo S.L. Slavkin H.C. Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 7254-7258Crossref PubMed Scopus (92) Google Scholar, 5Moradian-Oldak J. Matrix Biol. 2001; 20: 293-305Crossref PubMed Scopus (178) Google Scholar, 6Gibson C.W. Yuan Z.A. Hall B. Longenecker G. Chen E. Thyagarajan T. Sreenath T. Wright J.T. Decker S. Piddington R. Harrison G. Kulkarni A.B. J. Biol. Chem. 2001; 276: 31871-31875Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). In vitro amelogenin self-assembles into spherical structures, and this property depends on pH, ionic strength, and protein concentration (7Wiedemann-Bidlack F.B. Beniash E. Yamakoshi Y. Simmer J.P. Margolis H.C. J. Struct. Biol. 2007; 160: 57-69Crossref PubMed Scopus (66) Google Scholar). Using CD spectropolarimetry and NMR, we have reported that recombinant porcine amelogenin, under acidic conditions, can be classified as an intrinsically disordered or natively unfolded protein (IDP) 4The abbreviations used are: IDPintrinsically disordered proteinDLSdynamic light scatteringIDPintrinsically disordered proteinAIamelogenesis imperfectaANS8-anilinonaphthalene sulfonaterM180recombinant mouse amelogeninPMGpremolten globule-likeECMextracellular matrix proteinTRAPtyrosine-rich amelogenin polypeptide. (8Delak K. Harcup C. Lakshminarayanan R. Sun Z. Fan Y. Moradian-Oldak J. Evans J.S. Biochemistry. 2009; 48: 2272-2281Crossref PubMed Scopus (122) Google Scholar, 9Lakshminarayanan R. Il Y. Hegde B.G. Du C. Fan D. Moradian-Oldak J. Proteins. 2009; 76: 560-569Crossref PubMed Scopus (45) Google Scholar). In addition, we used various computational and experimental approaches to identify unfolded regions in amelogenin, further supporting the notion that it is a member of the IDP family (10Moradian-Oldak J. Lakshminarayanan R. Goldberg M. Amelogenins: Multifaceted Proteins for Dental and Bone Formation. Bentham Science Publishers Ltd., Dubai, U.A.E.2010: 106Google Scholar). Unlike folded proteins, IDPs lack regular secondary or tertiary structure and instead exist in an ensemble of conformations (9Lakshminarayanan R. Il Y. Hegde B.G. Du C. Fan D. Moradian-Oldak J. Proteins. 2009; 76: 560-569Crossref PubMed Scopus (45) Google Scholar). The resultant extended structure is thought to provide an increased surface area of interaction, the flexibility to interact with different partners, induced folding upon binding to partners, and accessibility to post-translational modifications (11Dunker A.K. Brown C.J. Lawson J.D. Iakoucheva L.M. Obradoviæ Z. Biochemistry. 2002; 41: 6573-6582Crossref PubMed Scopus (1457) Google Scholar, 12Dunker A.K. Silman I. Uversky V.N. Sussman J.L. Curr. Opin. Struct. Biol. 2008; 18: 756-764Crossref PubMed Scopus (773) Google Scholar, 13Mészáros B. Tompa P. Simon I. Dosztányi Z. J. Mol. Biol. 2007; 372: 549-561Crossref PubMed Scopus (223) Google Scholar). Notably, amelogenin exhibits properties that are hallmarks of IDPs, such as susceptibility to proteolysis, a high abundance of proline, and the ability to interact with other matrix components (8Delak K. Harcup C. Lakshminarayanan R. Sun Z. Fan Y. Moradian-Oldak J. Evans J.S. Biochemistry. 2009; 48: 2272-2281Crossref PubMed Scopus (122) Google Scholar, 14Fan D. Du C. Sun Z. Lakshminarayanan R. Moradian-Oldak J. J. Struct. Biol. 2009; 166: 88-94Crossref PubMed Scopus (29) Google Scholar). A number of acidic extracellular matrix proteins (ECM) associated with bone and tooth formation have also been characterized as being disordered (15Huq N.L. Cross K.J. Ung M. Reynolds E.C. Arch. Oral. Biol. 2005; 50: 599-609Crossref PubMed Scopus (90) Google Scholar, 16Fisher L.W. Torchia D.A. Fohr B. Young M.F. Fedarko N.S. Biochem. Biophys. Res. Commun. 2001; 280: 460-465Crossref PubMed Scopus (514) Google Scholar). The amino acid composition of amelogenin, however, differs from that of other ECM proteins. It lacks an appreciable amount of charged residues but contains higher amounts of hydrophobic residues (supplemental Fig. S1). Thus, in a charge-hydrophobicity plot amelogenin is placed at the border between the completely disordered and natively folded proteins (8Delak K. Harcup C. Lakshminarayanan R. Sun Z. Fan Y. Moradian-Oldak J. Evans J.S. Biochemistry. 2009; 48: 2272-2281Crossref PubMed Scopus (122) Google Scholar, 9Lakshminarayanan R. Il Y. Hegde B.G. Du C. Fan D. Moradian-Oldak J. Proteins. 2009; 76: 560-569Crossref PubMed Scopus (45) Google Scholar), whereas other ECM proteins completely fall in the disordered region. 5R. Lakshminarayanan and J. Moradian-Oldak, unpublished observations. Based on double wavelength CD plot spectra, we have defined porcine amelogenin in the subgroup of a “pre-molten globule” (PMG) state, meaning that it is not completely disordered but has residual secondary structure. Although a number of ECM proteins associated with bone and dentin have strong affinity for calcium, amelogenin is a relatively weak calcium binder but has a strong tendency to self-assemble (5Moradian-Oldak J. Matrix Biol. 2001; 20: 293-305Crossref PubMed Scopus (178) Google Scholar, 7Wiedemann-Bidlack F.B. Beniash E. Yamakoshi Y. Simmer J.P. Margolis H.C. J. Struct. Biol. 2007; 160: 57-69Crossref PubMed Scopus (66) Google Scholar, 9Lakshminarayanan R. Il Y. Hegde B.G. Du C. Fan D. Moradian-Oldak J. Proteins. 2009; 76: 560-569Crossref PubMed Scopus (45) Google Scholar). intrinsically disordered protein dynamic light scattering intrinsically disordered protein amelogenesis imperfecta 8-anilinonaphthalene sulfonate recombinant mouse amelogenin premolten globule-like extracellular matrix protein tyrosine-rich amelogenin polypeptide. Mutations observed in the AMELX, ENAM, KLK-4, and MMP-20 gene sequences lead to abnormal enamel formation and the diverse groups of genetically altered conditions known as amelogenesis imperfecta (18Wright J.T. Am. J. Med. Genet. A. 2006; 140: 2547-2555Crossref PubMed Scopus (105) Google Scholar, 19Wright J.T. Hart P.S. Aldred M.J. Seow K. Crawford P.J. Hong S.P. Gibson C.W. Hart T.C. Connect. Tissue Res. 2003; 44: 72-78Crossref PubMed Google Scholar, 20Lench N.J. Winter G.B. Hum. Mutat. 1995; 5: 251-259Crossref PubMed Scopus (92) Google Scholar, 21Hart P.S. Aldred M.J. Crawford P.J. Wright N.J. Hart T.C. Wright J.T. Arch. Oral Biol. 2002; 47: 261-265Crossref PubMed Scopus (74) Google Scholar, 22Mahoney E.K. Rohanizadeh R. Ismail F.S. Kilpatrick N.M. Swain M.V. Biomaterials. 2004; 25: 5091-5100Crossref PubMed Scopus (118) Google Scholar). Of the 23 mutations reported to date, 14 of the mutations are identified in the AMELX gene. These coding errors can disturb the secretion of amelogenin, produce mutations in the tyrosine-rich amelogenin polypeptide (TRAP) region, and cause truncation of amelogenin at the C terminus. All of these changes can contribute to defective enamel formation. Clinical manifestations of AI are heterogeneous and vary depending on the affected gene and location of the mutation. The enamel structure of the affected individuals is defined as either hypoplasia or hypomineralization or a combination of these (19Wright J.T. Hart P.S. Aldred M.J. Seow K. Crawford P.J. Hong S.P. Gibson C.W. Hart T.C. Connect. Tissue Res. 2003; 44: 72-78Crossref PubMed Google Scholar). Three missense mutations at the N terminus of human amelogenin involve substitution of threonine 21 to isoleucine (T21I), proline 40 to threonine (P40T), and histidine 47 to isoleucine (H47I). The T21I substitution in human amelogenin results in a phenotype described as hypomineralized/hypomatured enamel with brown discoloration (20Lench N.J. Winter G.B. Hum. Mutat. 1995; 5: 251-259Crossref PubMed Scopus (92) Google Scholar). The P40T mutation results in hypomaturation with a discolored phenotype (21Hart P.S. Aldred M.J. Crawford P.J. Wright N.J. Hart T.C. Wright J.T. Arch. Oral Biol. 2002; 47: 261-265Crossref PubMed Scopus (74) Google Scholar). The regular hierarchical prism structure observed in enamel is lacking the hypomineralized enamel, and the overall enamel shows porous and disoriented carbonated apatite crystals (22Mahoney E.K. Rohanizadeh R. Ismail F.S. Kilpatrick N.M. Swain M.V. Biomaterials. 2004; 25: 5091-5100Crossref PubMed Scopus (118) Google Scholar). It has been shown that the hypomineralized enamel exhibits poor hardness and elastic modulus compared with the unaffected enamel (23Xie Z. Swain M. Munroe P. Hoffman M. Biomaterials. 2008; 29: 2697-2703Crossref PubMed Scopus (53) Google Scholar, 24Xie Z.H. Mahoney E.K. Kilpatrick N.M. Swain M.V. Hoffman M. Acta Biomater. 2007; 3: 865-872Crossref PubMed Scopus (66) Google Scholar). As a result, the defective enamel fractures readily. The amino acid sequences of amelogenin across different vertebrates are highly conserved (25Toyosawa S. O'hUigin C. Figueroa F. Tichy H. Klein J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13056-13061Crossref PubMed Scopus (123) Google Scholar). Although the T21I point mutation from the human sequence occurs in an identical position in mouse amelogenin, the human P40T mutation occurs as Pro-41 in the mouse due to an additional methionine at residue 29 in the mouse (supplemental Fig. S1). Using recombinant mouse amelogenin and the AI mutants, Moradian-Oldak et al. (26Moradian-Oldak J. Paine M.L. Lei Y.P. Fincham A.G. Snead M.L. J. Struct. Biol. 2000; 131: 27-37Crossref PubMed Scopus (140) Google Scholar) have shown that the mutant amelogenins generated more heterogeneous assemblies than the wild type. It has been further reported that the interactions between wild type and AI variants are reduced by about 43% for the T21I mutant and 26% for the P41T mutants (27Paine M.L. Lei Y.P. Dickerson K. Snead M.L. J. Biol. Chem. 2002; 277: 17112-17116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Tanimoto et al. (28Tanimoto K. Le T. Zhu L. Witkowska H.E. Robinson S. Hall S. Hwang P. Denbesten P. Li W. J. Dent. Res. 2008; 87: 451-455Crossref PubMed Scopus (14) Google Scholar) have shown that the P41T mutation reduced the interaction of amelogenin with MMP-20, and the observed reduction in processing was purported to play an important role in the defective enamel formation. Despite these studies, details on the etiology of AI and the associated phenotypes remain largely unknown. Alterations in the self-assembly properties of amelogenin as a result of mutations are a clear manifestation of differences in enamel structure, and considerable insights can be gained by examining their physical and chemical properties. In this study, to gain more insight into the detailed secondary structures, degree of intrinsic disorder, and the changes in physico-chemical properties, we assessed the impact of T21I and P41T point mutations on recombinant full-length wild type (WT) mouse amelogenin (rM180) by circular dichroism, dynamic light scattering, and intrinsic tryptophan fluorescence techniques. We justify the use of recombinant proteins for the following reasons. 1) The differences in sequence between recombinant and native amelogenin are minimal and do not have a significant effect on protein self-assembly (26Moradian-Oldak J. Paine M.L. Lei Y.P. Fincham A.G. Snead M.L. J. Struct. Biol. 2000; 131: 27-37Crossref PubMed Scopus (140) Google Scholar, 29Simmer J.P. Lau E.C. Hu C.C. Aoba T. Lacey M. Nelson D. Zeichner-David M. Snead M.L. Slavkin H.C. Fincham A.G. Calcif. Tissue Int. 1994; 54: 312-319Crossref PubMed Scopus (163) Google Scholar). 2) Recombinant amelogenin has been demonstrated to be an excellent model for the study of amelogenin self-assembly (5Moradian-Oldak J. Matrix Biol. 2001; 20: 293-305Crossref PubMed Scopus (178) Google Scholar, 7Wiedemann-Bidlack F.B. Beniash E. Yamakoshi Y. Simmer J.P. Margolis H.C. J. Struct. Biol. 2007; 160: 57-69Crossref PubMed Scopus (66) Google Scholar, 30Fincham A.G. Moradian-Oldak J. Sarte P.E. Calcif. Tissue Int. 1994; 55: 398-400Crossref PubMed Scopus (50) Google Scholar). 3) Purification of native mouse amelogenin in substantial quantities free of contamination is difficult. Preparation of the recombinant His-tagged rM180 and the mutants have been described elsewhere (26Moradian-Oldak J. Paine M.L. Lei Y.P. Fincham A.G. Snead M.L. J. Struct. Biol. 2000; 131: 27-37Crossref PubMed Scopus (140) Google Scholar). The proteins were further purified on a Jupiter C4 semi-preparative reversed phase column (10 × 250 mm, 5 μm) Varian Prostar HPLC system (ProStar/Dynamics 6, version 6.41, Varian, Palo Alto, CA). A linear gradient of 60% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 2 ml/min was used. The amino acid sequence of mouse and human amelogenins shares ∼86% identity, and the two mutational spots are highly conserved (supplemental Fig. S1). Compared with the native mouse amelogenin sequence, all the proteins contain “RGHHHHHHGS” residues at the N terminus and lack a phosphorylation on Ser-16 (supplemental Fig. S2) (26Moradian-Oldak J. Paine M.L. Lei Y.P. Fincham A.G. Snead M.L. J. Struct. Biol. 2000; 131: 27-37Crossref PubMed Scopus (140) Google Scholar, 30Fincham A.G. Moradian-Oldak J. Sarte P.E. Calcif. Tissue Int. 1994; 55: 398-400Crossref PubMed Scopus (50) Google Scholar). CD spectra were obtained with a Jasco J-810 spectropolarimeter (A J-815 was used to perform the experiments displayed in supplemental Fig. S3) at a protein concentration of 0.4 mg/ml in 25 mm sodium acetate buffer, pH 5.8. Unless otherwise stated, the same concentration and buffer were used for all other experiments. The far UV-CD spectra were recorded using a 0.1-cm path length cell under constant nitrogen flush with a step size of 0.1 nm, bandwidth of 2 nm, and an averaging time of 3 s. The final spectra reported were an average of 16 scans. All the spectra were background-subtracted and smoothed by the Savitzky-Golay method using a window size of 5 nm (no smoothing was performed for any of the scans performed by the J-815). For the variable temperature CD spectra, an external thermostat was used that controlled the temperature to within 0.1 °C. The thermal denaturation of proteins was determined by monitoring the changes in CD intensity at 200 and 224 nm for all the proteins. The protein solutions were heated from 10 to 70 °C and then cooled back to 10 °C at a rate of 5 °C/min. For the thermal stability measurements, the data obtained from CD intensity at 200 nm were fit into a two-state model using the Origin 7.5 software. The thermodynamic parameters were determined by the procedure reported by Pace and Scholtz (31Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure: A Practical Approach. IRL Press, Oxford, UK1997: 299-321Google Scholar). For the refolding kinetics, protein solutions (0.4 mg/ml) were heated to 70 °C and kept at that temperature for 5 min before being cooled to 25 °C. Changes in CD intensity at 200 nm as a function of time were then monitored. We noted that rM180 and P41T rapidly returned to equilibrium. We therefore monitored the changes from the denaturation temperature onward. The samples reached 25 °C in 70 s, and the data were fit into a single exponential function using Origin 7.5 software. Intrinsic tryptophan fluorescence spectra were recorded on a QuantaMaster QM-4SE spectrofluorometer (Photon Technology International). The protein solution (0.4 mg/ml, pH 5.8, sodium acetate buffer (25 mm), 25 °C) was excited at 290 nm, and the emission spectra were monitored between 300 and 400 nm with a step size of 1 nm, using a 10-mm path length cell. For variable temperature measurements, the Peltier controller was used. The temperature scan was performed at 5 °C intervals from 25 to 70 °C. Measurements were made at each temperature after 5 min to allow the sample to equilibrate in the cell. 8-Anilinonaphthalene sulfonate (ANS) fluorescence was recorded after excitation of the dye or dye-protein complexes at 350 nm. The emission spectra were monitored between 400 and 600 nm. Measurements of the hydrodynamic radii of the various amelogenin proteins (0.4 mg/ml, pH 5.8, 25 mm sodium acetate buffer) were performed using a Wyatt DynaPro Nanostar dynamic light scattering instrument (Wyatt Technology, Santa Barbara, CA). For room temperature experiments, the temperature was set to 25 °C, although variable temperature experiments were performed between 20 and 60 °C at a ramp rate of 0.25 °C/min. The data were analyzed using Dynamics 7.0 software. The dynamic light scattering data were produced by the program performing a regularization fit using the Dynals algorithm on the resultant autocorrelation functions. A Rayleigh sphere model was used for the analysis, meaning that the hydrodynamic radii calculated were sphere-equivalent radii. By measuring the fluctuations in the laser light intensity scattered by the sample, the instrument was able to detect the speed (diffusion coefficient) at which the particles were moving through the medium. This value is converted to hydrodynamic radius using the Stokes-Einstein relation shown in Equation 1,D=kT6πηRH(Eq. 1) where D is the is the diffusion coefficient; k is the Boltzmann constant; T is the absolute temperature; η is the viscosity, and RH is the sphere-equivalent hydrodynamic radius (32Schärtl W. Light Scattering from Polymer Solutions and Nanoparticle Dispersions. 1st Ed. Springer-Verlag, Heidelberg, Germany2007Google Scholar). The secondary structures of WT rM180 and mutant amelogenins were recorded in 25 mm sodium acetate buffer, pH 5.8. At 25 °C, WT and mutant amelogenins all exhibited a strong negative minimum around 202 nm, which is characteristic of intrinsically disordered proteins (Fig. 1A). For the T21I mutant, CD spectra showed a similar minimum around 202 nm, but the CD intensity was much lower than the other two proteins. Steady state tryptophan fluorescence is a useful technique to probe the environment around the aromatic amino acid residues (33Lakowicz J.R. Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum Press, New York1999Crossref Google Scholar, 34Shen C. Menon R. Das D. Bansal N. Nahar N. Guduru N. Jaegle S. Peckham J. Reshetnyak Y.K. Proteins. 2008; 71: 1744-1754Crossref PubMed Scopus (31) Google Scholar). Illuminating proteins with 290-nm excitation wavelength selectively excites tryptophan. Fig. 1B shows the plot of normalized emission intensity versus emission wavelength for all three amelogenins at 25 °C. Both the WT and the P41T mutant had an emission maximum around 346 nm, a characteristic feature of “class III” proteins that have tryptophan residues exposed to mobile water molecules (34Shen C. Menon R. Das D. Bansal N. Nahar N. Guduru N. Jaegle S. Peckham J. Reshetnyak Y.K. Proteins. 2008; 71: 1744-1754Crossref PubMed Scopus (31) Google Scholar). However, for the T21I mutant, the maximum was blue-shifted to 338 nm (characteristic of “class II” proteins) indicating partially buried nature of the fluorophores. DLS allows the measurement of hydrodynamic radii of proteins. DLS studies revealed that WT rM180 and the P41T mutant had hydrodynamic radii of 3.4 and 3.5 nm, respectively (Table 1), which are smaller than expected for a completely extended conformation (4.2 nm) and larger than for a globular conformation (2.2 nm) of proteins with the same amino acid chain lengths (35Uversky V.N. Eur. J. Biochem. 2002; 269: 2-12Crossref PubMed Scopus (803) Google Scholar). Based on the analysis of CD spectra and hydrodynamic dimensions, Uversky classified IDPs into intrinsic coil-like and intrinsic premolten globule-like (PMG) proteins (36Uversky V.N. Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1478) Google Scholar). The two subclasses can be distinguished by the relationship between increases in hydrodynamic radii and molecular weight (36Uversky V.N. Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1478) Google Scholar). Fig. 1C compares the hydrodynamic radii of PMG-like proteins with that of rM180 and P41T mutant. The hydrodynamic radii for the two proteins have an excellent agreement with the dimensions of PMG-like proteins. To further confirm that rM180 and P41T belong to the PMG-like subclass, ANS binding assays were recorded (Fig. 1D). Excitation of ANS at 350 nm had an emission maximum at 505 nm in acetate buffer, pH 5.8. However, when added to rM180 or P41T mutant, the fluorescence maximum was blue-shifted to 475 nm, and an 18–20-fold increase in intensity of ANS was observed in comparison with the emission in the absence of rM180 or P41T (Fig. 1D). Thus, ANS fluorescence and DLS measurements confirm that at pH 5.8 and 25 °C, rM180 and the P41T mutant are monomeric and more compact (PMG-like) than completely extended IDPs. Interestingly, at the same pH and protein concentration, the T21I mutant existed as an oligomer with a hydrodynamic radius of 7 nm (Table 1).TABLE 1Hydrodynamic radii and polydispersity values (% PD) of rM180 and the mutants at 25 °C, pH 5.8, as determined by DLS analysisProteinRHMassPDnm%%rM1803.41 ± 0.1597.04 ± 1.0924.29 ± 10.97T21I7.07 ± 0.2398.81 ± 1.5234.6 ± 9.81P41T3.52 ± 0.1198.98 ± 1.0641.97 ± 12.19 Open table in a new tab A hallmark of IDPs is that the heat-induced conformational transition is reversible (37Uversky V.N. Protein J. 2009; 28: 305-325Crossref PubMed Scopus (227) Google Scholar). To infer the effect of temperature on WT rM180 and the two AI mutants, the thermal denaturation/refolding behavior of amelogenins was examined by monitoring the CD intensity at 224 nm as a function of temperature. Fig. 2, A–C, show the changes in CD intensity at 224 nm as a function of temperature for the three proteins. Unlike the linear thermal transition observed for recombinant porcine amelogenin and other IDPs, rM180 exhibited a biphasic behavior (Fig. 2A) (8Delak K. Harcup C. Lakshminarayanan R. Sun Z. Fan Y. Moradian-Oldak J. Evans J.S. Biochemistry. 2009; 48: 2272-2281Crossref PubMed Scopus (122) Google Scholar, 9Lakshminarayanan R. Il Y. Hegde B.G. Du C. Fan D. Moradian-Oldak J. Proteins. 2009; 76: 560-569Crossref PubMed Scopus (45) Google Scholar). At low temperatures, the CD intensity increased with temperature indicating a noncooperative unfolded-to-folded transition. Above 45 °C, denaturation began with a concomitant reduction in CD intensity (indicated by black arrow in Fig. 2A), as has been observed for folded proteins (38Hill R.B. DeGrado W.F. Structure. 2000; 8: 471-479Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). On cooling (open circles), the CD intensity retraced back slowly, and refolding began at 50 °C (indicated by red arrow in Fig. 2A). Complete refolding occurred at around 41 °C (indicated by blue arrow in Fig. 2A), and the CD intensity followed the same path as the heating cycle upon further cooling. Thus, WT rM180 exhibited a weak hysteresis upon denaturation (heating) and refolding (cooling). While heating the T21I mutant, the CD intensity increased at a similar rate, but it decreased much more sharply above 45 °C and reached a plateau at higher temperatures (Fig. 2B). On cooling, the intensity retraced more slowly than rM180 and refolding began below 42 °C (indicated by red arrow in Fig. 2B), and the process was completed at 33 °C (indicated by blue arrow in Fig. 2B). Thus, the T21I mutant exhibited a larger hysteresis deflection than was observed for rM180. The denaturation of the P41T mutant followed the same path as rM180 (Fig. 2C). The onset of refolding, however, occurred around 45 °C (indicated by red arrow in Fig. 2C), but complete refolding was achieved only below 30 °C (indicated by blue arrow in Fig. 2B). The loss in CD intensity at elevated temperatures suggests that rM180 and the mutants denature to an aggregated state. To further understand the thermal behavior, all three proteins were subjected to multiple heating/cooling cycles and monitored as before (supplemental Fig. S3). For rM180, the CD intensity patterns derived from various cycles could be superimposed. However, both mutants showed considerable lag after the 1st heating cycle (supplemental Fig. S3). The presence of a hysteresis during thermal denaturation implies that unfolding/refolding takes a different path and varies for the two mutants compared with WT rM180. For example, during refolding hardly any original conformation was detectable at 37 °C for the two mutants, whereas rM180 reaches the original conformation below 42 °C. Therefore, to assess the influence of mutations on the refolding ability, the proteins were completely denatured by heating to 70 °C and cooled rapidly to 25 °C. The change in ellipticity values at 200 nm was monitored over time. Fig. 2D shows the kinetics of refolding for WT and the mutant proteins. For all the proteins, a large change in signal occurred within the first few seconds. This burst phase was accompanied by a slower observable phase. F" @default.
• W2036931844 created "2016-06-24" @default.
• W2036931844 creator A5030235578 @default.
• W2036931844 creator A5035157534 @default.
• W2036931844 creator A5043108596 @default.
• W2036931844 creator A5055824674 @default.
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• W2036931844 date "2010-12-01" @default.
• W2036931844 modified "2023-09-28" @default.
• W2036931844 title "Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins" @default.
• W2036931844 cites W1575285231 @default.
• W2036931844 cites W1720678970 @default.
• W2036931844 cites W1829746528 @default.
• W2036931844 cites W1931164952 @default.
• W2036931844 cites W1964839527 @default.
• W2036931844 cites W1968242343 @default.
• W2036931844 cites W1968826334 @default.
• W2036931844 cites W1969831606 @default.
• W2036931844 cites W1974525618 @default.
• W2036931844 cites W1976203135 @default.
• W2036931844 cites W1978150593 @default.
• W2036931844 cites W1985764073 @default.
• W2036931844 cites W1991865349 @default.
• W2036931844 cites W1994633856 @default.
• W2036931844 cites W2002147692 @default.
• W2036931844 cites W2003813143 @default.
• W2036931844 cites W2004314780 @default.
• W2036931844 cites W2013846682 @default.
• W2036931844 cites W2019511236 @default.
• W2036931844 cites W2025925640 @default.
• W2036931844 cites W2029487298 @default.
• W2036931844 cites W2030710208 @default.
• W2036931844 cites W2039397725 @default.
• W2036931844 cites W2043833442 @default.
• W2036931844 cites W2049636057 @default.
• W2036931844 cites W2050446363 @default.
• W2036931844 cites W2052677774 @default.
• W2036931844 cites W2053399901 @default.
• W2036931844 cites W2056359726 @default.
• W2036931844 cites W2058040839 @default.
• W2036931844 cites W2066895299 @default.
• W2036931844 cites W2070792168 @default.
• W2036931844 cites W2073745520 @default.
• W2036931844 cites W2079438735 @default.
• W2036931844 cites W2081810556 @default.
• W2036931844 cites W2082584989 @default.
• W2036931844 cites W2084333983 @default.
• W2036931844 cites W2085704045 @default.
• W2036931844 cites W2086026048 @default.
• W2036931844 cites W2094130673 @default.
• W2036931844 cites W2096492470 @default.
• W2036931844 cites W2097891553 @default.
• W2036931844 cites W2109567227 @default.
• W2036931844 cites W2111250324 @default.
• W2036931844 cites W2112189322 @default.
• W2036931844 cites W2112629628 @default.
• W2036931844 cites W2112761293 @default.
• W2036931844 cites W2117695722 @default.
• W2036931844 cites W2119693327 @default.
• W2036931844 cites W2122248707 @default.
• W2036931844 cites W2124220488 @default.
• W2036931844 cites W2125591567 @default.
• W2036931844 cites W2132620042 @default.
• W2036931844 cites W2134370487 @default.
• W2036931844 cites W2143234288 @default.
• W2036931844 cites W2144697028 @default.
• W2036931844 cites W2151796887 @default.
• W2036931844 cites W2163828149 @default.
• W2036931844 cites W2164473331 @default.
• W2036931844 cites W2165186407 @default.
• W2036931844 cites W2166899489 @default.
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