Modular Motif, Structural Folds and Affinity Profiles of the PEVK Segment of Human Fetal Skeletal Muscle Titin
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m008851200
ISSN1083-351X
AutoresGustavo Gutierrez-Cruz, Ann H. Van Heerden, Kuan Wang,
Tópico(s)Nuclear Structure and Function
ResumoThe extension of the PEVK segment of the giant elastic protein titin is a key event in the elastic response of striated muscle to passive stretch. PEVK behaves mechanically as an entropic spring and is thought to be a random coil. cDNA sequencing of human fetal skeletal PEVK reveals a modular motif with tandem repeats of modules averaging 28 residues and with superrepeats of seven modules. Conformational studies of bacterially expressed 53-kDa fragment (TP1) by circular dichroism suggest that this soluble protein contains substantial polyproline II (PPII) type left-handed helices. Urea and thermal titrations cause gradual and reversible decrease in PPII content. The absence of sharp melting in urea and thermal titrations suggests that there is no long range cooperativity among the PPII helices. Studies with solid phase and surface plasmon resonance assays indicate that TP1 interacts with actin and some but not all cloned nebulin fragments with high affinity. Interestingly, Ca2+/calmodulin and Ca2+/S100 abolish nebulin/PEVK interaction. We suggest that in aqueous solution, PEVK is an open and flexible chain of relatively stable structural folds of the polyproline II type. PEVK region of titin may be involved in interfilament association with thin filaments in a calcium/calmodulin-sensitive manner. This adhesion may modulate titin extensibility and elasticity. The extension of the PEVK segment of the giant elastic protein titin is a key event in the elastic response of striated muscle to passive stretch. PEVK behaves mechanically as an entropic spring and is thought to be a random coil. cDNA sequencing of human fetal skeletal PEVK reveals a modular motif with tandem repeats of modules averaging 28 residues and with superrepeats of seven modules. Conformational studies of bacterially expressed 53-kDa fragment (TP1) by circular dichroism suggest that this soluble protein contains substantial polyproline II (PPII) type left-handed helices. Urea and thermal titrations cause gradual and reversible decrease in PPII content. The absence of sharp melting in urea and thermal titrations suggests that there is no long range cooperativity among the PPII helices. Studies with solid phase and surface plasmon resonance assays indicate that TP1 interacts with actin and some but not all cloned nebulin fragments with high affinity. Interestingly, Ca2+/calmodulin and Ca2+/S100 abolish nebulin/PEVK interaction. We suggest that in aqueous solution, PEVK is an open and flexible chain of relatively stable structural folds of the polyproline II type. PEVK region of titin may be involved in interfilament association with thin filaments in a calcium/calmodulin-sensitive manner. This adhesion may modulate titin extensibility and elasticity. kilobase pair dithiothreitol enzyme-linked immunosorbent assay polyproline II 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; N-hydroxysulfosuccinimide. The monumental sequencing work of Labeit and Kolmerer (1Labeit S. Kolmerer B. Science. 1995; 270: 293-296Crossref PubMed Scopus (974) Google Scholar) has revealed the complete domain organization of the giant elastic protein titin. The bulk of cardiac titin consists of predominately two types of sequence motifs: immunoglobulin (Ig) and fibronectin arranged in three levels of motifs (repeats, superrepeats, and segments). In addition to these well characterized domains, a novel motif consisting of mainly four amino acid residues, Pro, Glu, Val, and Lys, is discovered in the elastic I band region of titin. The length of this PEVK segment varies among muscles, ranging from 183 residues in the human cardiac titin to 2174 residues in human soleus skeletal muscle. Differential splicing of the titin transcripts in the PEVK region as well as in an adjacent tandem Ig segment near the A band produce these size isoforms of titin (1Labeit S. Kolmerer B. Science. 1995; 270: 293-296Crossref PubMed Scopus (974) Google Scholar, 2Freiburg A. Trombitas K. Hell W. Cazorla O. Fougerousse F. Centner T. Kolmerer B. Witt C. Beckmann J.S. Gregorio C.C. Granzier H. Labeit S. Circ. Res. 2000; 86: 1114-1121Crossref PubMed Scopus (272) Google Scholar). Since the selective expression of titin size isoforms imparts distinct elasticity to skeletal and cardiac muscles, with the muscle expressing longer titin being more compliant (3Wang K. McCarter R. Wright J. Beverly J. Ramirez-Mitchell R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7101-7105Crossref PubMed Scopus (247) Google Scholar), the observed length variation of PEVK and tandem Igs in titin isoforms immediately suggests the possibility that either or both segments may be the elastic elements. Labeit and Kolmerer (1Labeit S. Kolmerer B. Science. 1995; 270: 293-296Crossref PubMed Scopus (974) Google Scholar) speculated that the PEVK region, with a predicted nonfolded polypeptide, is the key elastic element of titin. The concept of reversible unfolding and folding of Ig domains was considered unlikely, on the ground of the thermodynamic stability of Ig domains (4Pastore A. Politou A.S. Thomas D.J. J. Muscle Res. Cell Motil. 1996; 17: 114-115Google Scholar, 5MuhleGoll C. Nilges M. Pastore A. J. Biomol. NMR. 1997; 9: 2-10Crossref PubMed Scopus (10) Google Scholar). Recent works on the elasticity of single titin molecules (6Kellermayer M.S.Z. Smith S.B. Granzier H.L. Bustamante C. Science. 1997; 276: 1112-1116Crossref PubMed Scopus (1010) Google Scholar), single myofibrils (7Linke W.A. Ivemeyer M. Olivieri N. Kolmerer B. Ruegg J.C. Labeit S. J. Mol. Biol. 1996; 261: 62-71Crossref PubMed Scopus (225) Google Scholar), and single fibers (8Wang K. McCarter R. Wright J. Beverly J. Ramirez-Mitchell R. Biophys. J. 1993; 64: 1161-1177Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 9Granzier H.L.M. Wang K. Biophys. J. 1993; 65: 2141-2159Abstract Full Text PDF PubMed Scopus (106) Google Scholar) revealed that, stretched modestly, sarcomere elasticity can be explained by the straightening of tandem Ig segment (without unfolding), followed by the extension of a permanently unfolded PEVK segment (10Linke W.A. Granzier H. Biophys. J. 1998; 75: 2613-2614Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To further evaluate molecular theories of titin elasticity, studies of the conformation and stability of different classes of titin domains are essential. The systematic NMR studies of expressed single Ig and fibronectin domains by Pastore and coworkers (11Politou A.S. Gautel M. Pfuhl M. Labeit S. Pastore A. Biochemistry. 1994; 33: 4730-4737Crossref PubMed Scopus (70) Google Scholar) have provided insightful structural details and a possible model of force generation. In contrast, no molecular characteristics of the PEVK region have yet been described. We report the initial sequence and molecular analysis of a proline-rich region that we identified independently by screening expression libraries of human fetal skeletal muscle with anti-titin antibodies. One 2.5-kb1 clone was found to consist of tandem repeats of a fundamental module that averages 28 residues, mostly of PEVK residues. This sequence is classified as a PEVK segment by its high homology with human soleus PEVK. Conformational studies of a PEVK fragment (designated as TP1, containing 16 PEVK modules, 468 amino acid residues) indicate that PEVK is an open and flexible chain with stable structural folds, perhaps of left-handed polyproline II helices, but contains little, if any, α-helix and β-sheet types of secondary structure. Protein interaction studies with solid phase assays reveal modest micromolar range interactions between TP1 and nebulin and actin. Our data suggest that the PEVK region may serve multiple functions. It may serve as an entropic spring of a chain of structural folds. The PEVK region also may be a site of interaction with other myofilament proteins to form interfilament connectivity in the sarcomere. A λ gt11 human fetal skeletal muscle library was screened by Western blot with a goat anti-rabbit skeletal muscle titin (goat 812) that has been absorbed with E. coli blots and supplemented with a mixture of monoclonal antibodies (RT10, -11, -13, and -15) (8Wang K. McCarter R. Wright J. Beverly J. Ramirez-Mitchell R. Biophys. J. 1993; 64: 1161-1177Abstract Full Text PDF PubMed Scopus (197) Google Scholar). One clone, 5-1-2, containing a 2.5-kb open reading frame (designated as hfT11; GenBankTM accession number AF 321609) was subcloned into Bluescript by ECoR1 digestion and ligation. Nested set deletions were produced by exoIII digestion, and each clone was sequenced by Sequence/T7 polymerase/universal and reverse primer with the dideoxy method. The 7-deaza reaction was run to clear up compression. The DNA sequence and deduced amino acid sequence were analyzed by MacVector and MACAW for homology and repeats (12Wang K. Knipfer M. Huang Q.Q. VanHeerden A. Gutierrez G. Quian X. Stedman H. J. Biol. Chem. 1996; 271: 4114-4304Google Scholar). A 1.4-kb open reading frame derived from hfT11 was subcloned into a pET3d plasmid by digesting Bluescript clone with HinfI and ligated to pET 3d, essentially as described in (12Wang K. Knipfer M. Huang Q.Q. VanHeerden A. Gutierrez G. Quian X. Stedman H. J. Biol. Chem. 1996; 271: 4114-4304Google Scholar). The expressed protein, TP1 (51,467,469 residues), was purified from a 4-liter culture of BL21(DE3)pLysS host cells transformed with pET3d HinfI plasmid that was incubated at 37 °C for 3.5 h upon isopropyl-1-thio-β-d-galactopyranoside induction (0.4 mm isopropyl-1-thio-β-d-galactopyranoside when A 550 = 0.6–0.8). The bacteria were collected by centrifugation at 3800 × g for 10 min and lysed in a French press cell (3 × 1500 p.s.i.) in 45 ml of lysis buffer (10 mm NaPi, 1 mm EDTA, 0.1 mm DTT, 10 μg/ml leupeptin, 1 mg/ml casein, 1 mm diisopropyl fluorophosphate, 0.5 mmphenylmethylsulfonyl fluoride, pH 7.0), followed by centrifugation at 12,000 × g for 20 min at 4 °C. The supernatant was dialyzed overnight against (10 mm NaPi, 1 mmEDTA, 0.1 mm DTT, 0.5 mm phenylmethylsulfonyl fluoride, pH 7.0) at 4 °C, clarified by centrifugation at 27,000 × g for 20 min, and loaded to a Whatman phosphocellulose P-11 column (5.2 × 4.0 cm) equilibrated in the same buffer at 4 °C. TP1 was eluted between 0.4 and 0.5m NaCl by a linear gradient (0–1 m NaCl). Pooled fractions containing TP1 were dialyzed overnight against 20 mm NaPi, 1 mm EDTA, 0.1 mm DTT, pH 7.0, at 4 °C and applied to a Source 15S PE column (Amersham Pharmacia Biotech) equilibrated in the same buffer at 4 °C. Elution with a linear NaCl gradient (0–0.3 m) yielded ∼95% pure TP1 between 0.10 and 0.12 m NaCl (15 mg/4-liter culture). Polyacrylamide gel electrophoresis was done in 12% Laemmli gel. Western blot was done with electrophoretic transfer on nitrocellulose membrane (S&S BA85) in a Bio-Rad semidry electroblotter at 1.5 mA/cm2 for 60 min using a buffer system modified from Khyse-Andersen (13Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2143) Google Scholar) Briefly, the blotting assembly was set on the anode electrode plate of a semidry transfer unit (Bio-Rad) in the following order: two sheets of filter paper (GB002; Schleicher & Schüll) presoaked in anode buffer 1 (300 mm Tris-Cl, 0.05% SDS, 10% methanol, 10 mmβ-mercaptoethanol, pH 10.4); nitrocellulose membrane presoaked in anode buffer 2 (25 mm Tris-Cl, 0.05% SDS, 10% methanol, 10 mm β-mercaptoethanol, pH 10.4); SDS gels presoaked in the cathode buffer (25 mm Tris-Cl, 0.05% SDS, 10% methanol, 10 mm β-mercaptoethanol, and 40 mmamino-N-hexanoic acid, pH 10.4) for 15 s; and four sheets of filter paper presoaked in the cathode buffer. Transfer of proteins was performed at 1.5 mA/cm2 of gel for 60 min. Blots were stained with anti-titin monoclonal antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (rabbit anti-mouse IgG, IgM, and IgA (H + L)). Monoclonal antibodies against rabbit skeletal titin were prepared previously in this laboratory by standard procedures. Monoclonal antibody RT11, an IgM, was found to react with the PEVK segment and was used to monitor the expression and purification of TP1 by Western blots. The concentration of TP1 was determined spectroscopically with a calculated extinction coefficient at 280 nm of 0.025 for 1 mg/ml. Stoke's radius of TP1 was estimated by the method of Laurent and Killander (14Laurent T.C. Killander J. J. Chromatogr. 1964; 14: 317-330Crossref Google Scholar). A 10 × 300-mm Superose 6 HR (Amersham Pharmacia Biotech) equilibrated with buffer I (10 mm imidazole, 100 mm KCl, 1 mm CaCl2, 1 mm MgCl2, pH 7.0) was calibrated by determining the elution volume (V e) of a set of standard proteins of known Stoke's radii: human fibrinogen (107 Å), bovine thyroglobulin (85 Å), horse spleen ferritin (61 Å), bovine liver catalase (52.2 Å), rabbit muscle aldolase (48.1Å), bovine serum albumin (35.5 Å), ovalbumin (30.5 Å), bovine pancreas chymotrypsinogen A (20.9 Å), and bovine pancreas ribonuclease A (16.4 Å). The void volume (V o) was determined using blue dextran. The Stoke's radius of TP1 was determined from the calibration curve obtained by the linear fitting of Stoke's radius versus(-logKav)1/2 CD spectra of TP1 and poly-l-proline (M r 5000; Sigma P-2254) in 10 mm potassium phosphate buffer, pH 7.0, were recorded on a JASCO-J-600 spectropolarimeter (Easton, MD) at seven temperatures (2, 10, 20, 30, 40, 50, and 70 °C) under a constant nitrogen flow at 9 liters/min to flush away oxygen that absorbs below 200 nm. The instrument was calibrated with a 0.06% aqueous solution of ammonium d-10-camphosulfonate in the near UV region and with a 0.015% aqueous solution of d-(−)-pantolactone (Aldrich, WI) in the far UV region. A quartz cuvette (Hellma, Plainview, NY) with a 0.01-cm light path was used. For urea titration at 20 °C, TP1 (22 mg/ml) or polyproline (2 mg/ml) was dialyzed against various concentrations of urea (2, 4, 6, and 8 m) in the same buffer. Concentrated urea solutions were prepared in water by weight using the density data of Kanahara (15Kawahara K. Tanford C. J. Biol. Chem. 1966; 241: 3228-3232Abstract Full Text PDF PubMed Google Scholar) and then adjusted to desired concentration with water and 10× concentrated buffer. Thermal denaturation of TP1 (2.2 mg/ml) and polyproline (0.25 mg/ml) was monitored by following the change in CD at 205 nm (TP1) or 201 nm (polyproline) from 20 to 90 °C at a rate of 50 °C/h using a JASCO model PTC-348W Peltier type thermoelectric control system and a demountable 0.01-cm path length rectangular cuvette. Purified TP1 (220 μg/ml in 20 mm sodium phosphate, 1 mm EDTA, 150 mm NaCl, pH 7.0) was absorbed onto microtiter plates overnight at 4 °C, washed once with TBS-T (10 mmTris-Cl, pH 7.5, 150 mm NaCl, 0.05% (v/v) Tween 20), and blocked with 0.02% (w/v) bovine serum albumin in TBS-T for 1 h at 37 °C, followed by incubation with actin and nebulin fragments (NA3, NA4, NC17, ND8, ND66) in buffer I (10 mm imidazole, 150 mm KCl, 1 mm CaCl2, 2 mm MgCl2, pH 7.0) at a concentration ranging from 0.7 to 50 μg/ml for 2 h at 37 °C. After washing plates three times with TBS-T, mouse monoclonal antibodies against actin (JL20), NA3 (N109), NA4 (N103), NC17 (N107), ND8 (N113), and ND66 (N113) were incubated for 1 h at 37 °C in 0.2% bovine serum albumin-TBS-T. Plates were washed three times with TBS-T and then incubated with a peroxidase-conjugated rabbit anti-mouse antibody for 1 h. at 37 °C in bovine serum albumin-TBS-T, followed by five washes with TBS-T. Color was developed for 20 min at 25 °C by adding ABTS-H2O2 substrate, 10 μl of H2O2, 10 ml of 100 mm citrate buffer, pH 4.2. Absorbance at 405 nm was measured using an ELISA microtiter plate reader (12Wang K. Knipfer M. Huang Q.Q. VanHeerden A. Gutierrez G. Quian X. Stedman H. J. Biol. Chem. 1996; 271: 4114-4304Google Scholar, 16Jin J.P. Wang K. J. Biol. Chem. 1991; 266: 21215-21223Abstract Full Text PDF PubMed Google Scholar). Surface plasmon resonance assays were done using a real time biosensor, IAsys Manual System (Affinity Sensors, Cambridge, UK). TP1 was attached to caboxymethylated dextran on the cuvette surface via EDC/NHS. The cuvette was activated with 0.4 m EDC, 0.1 mNHS in water for 8 min, washed twice with phosphate-buffered saline with 0.05% Tween 20, followed by a wash with 10 mmacetate buffer pH 5.0 for 3 min, and incubated with PT1 (0.22 mg/ml in 10 mm acetate buffer, pH 5.0) for 10 min. The cuvette was washed twice with phosphate-buffered saline with 0.05% Tween 20, followed by 1 m ethylenediamine for 3 min to block excess activated carboxyl groups. The cuvette was then washed with acetate buffer (3×), 10 mm HCl, acetate buffer (3×), and finally PBST (3×). A blank cuvette from the same lot was processed in parallel, without TP1. The use of ethylenediamine to block excess carboxyls is essential for the study of the interaction of TP1 with the highly basic nebulin fragments. The use of hydroxylamine, as recommended by the manufacturer, led to unacceptably high binding of basic proteins to the control blank cuvette even at high ionic strength (200 mm KCl). The introduction of basic groups to the dextran led to a gel layer that showed no significant electrostatic binding of NA4 (pI = 9.24) at and above 100 mm KCl (with response <50 arc seconds). The binding of nebulin fragments (NA3, NA4, NA29, NC17, and ND66), actin, troponin, tropomyosin, calmodulin (bovine brain), and S100αα (Sigma) to immobilized TP1 was done at 1 μm in 100 mm KCl, 1 mm CaCl2, 2 mm MgCl2, 10 mm imidazole, pH 7.0, at 25 °C. For some solutions, EGTA (1 mm) was present to lower pCa to 8.0. Protein binding to the gel layer alters the refractive index profile occurring within the evanescent field and changes the measured resonance angle of the intensity peak (in arc seconds). The change in arc seconds is assumed to be proportional to the amount of bound substance (17Stenberg E. Persson B. Roos H. Urbaniczky C. J. Colloid Interface Sci. 1991; 143: 513-526Crossref Scopus (998) Google Scholar). The association kinetics was followed for 10–15 min prior to washing with the interaction buffer to follow the time course of dissociation. At the end of each experiment, the cuvette was washed with 10 mm HCl for 1 min and buffer I (3×) to regenerate the cuvette. All of the data reported here were obtained on the same cuvette within a 2-day period to facilitate comparison. Resonance angles of the treated control cuvette were measured in parallel and subtracted from the corresponding values obtained with the TP1 cuvette. Localization of epitopes of monoclonal antibodies RT11, RT13, and RT15 in mechanically split rabbit psoas muscle fibers was done as described previously (18Wright J. Huang Q.Q. Wang K. J. Muscle Res. Cell Motil. 1993; 14: 476-483Crossref PubMed Scopus (84) Google Scholar). Briefly, the split muscle fibers from rabbit psoas muscle in relaxing buffer were hand-stretched to various degrees and attached to gold single-slot grid, fixed in 3.7% formaldehyde, labeled with anti-titin supernatants, and followed by rabbit anti-mouse immunoglobulin and then protein A gold bead (5 nm) conjugates. The labeled fibers were fixed in glutaraldehyde before embedded in Epon-Araldite, sectioned, and stained with uranyl acetate and Reynold's lead citrate for observation in an electron microscope (JEOL 100CX). Epitope translocation was determined by plotting the center to center distance from each epitope to the Z line or to the M line as a function of sarcomere length. Sequence analysis of a 2.5-kb cDNA clone (5-1-2) obtained by screening a human fetal muscle cDNA library with anti-titin antibodies revealed an open reading frame (designated as hfT11) of 786 residues that is enriched in prolines, glutamates, valines, and lysines (25% Pro, 16% Glu, 15% Val, 16% Lys) (GenBankTM accession number AF 321609). A search for internal sequence homology by matrix plots with MacVector indicates substantial internal repeats (Fig. 2 A). Further analysis with MACAW to search for sequence homology alignment revealed that the majority of the sequence near the C-terminal side consists of 18 tandem repeats of sequence modules of an average of 28 residues (ranging from 25 to 30). The alignment of these modules and nonrepeat sequences in Fig. 1 showed the consensus of PE(V/A)PKEVVPEKK(A/V) PVAPPKKPE(V/A)PPVKV. The variability in length among these modules occurs between the first and second prolines, between the EKK and PP and between PP and VKV (in length). Closer examination of these repeats further revealed the presence of adjoining superrepeats from HRT14–20 (designated as SRA) and HRT 21–27 (designated as SRB). Within each superrepeat, seven modules are arranged in the same order of abcdefg types of module. The N-terminal side of this open reading frame is more variable. Four modules (HRT1–4) with low similarity are found. The next four modules, HRT10–13, show little homology with the modules from HRT14–27, but each module has ∼25–35 residues, and three show a PE at the beginning of the module. These four modules are classified as nonrepeats. This nonrepeat region is characterized by clusters of acidic residues (e.g. HRT4) and basic residues (KRRRK in HRT8). The matrix plot of hfT11 against human soleus PEVK indicates that hfT11 open reading frame sequence shares significant homology with the C-terminal side of the PEVK region of human soleus titin (Fig.2 A).Figure 1Partial sequence of PEVK segment of human fetal skeletal muscle titin. A 2.5-kb cDNA clone (hfT11) was obtained by screening a human fetal muscle library with a mixture of polyclonal and monoclonal antibodies to titin. The deduced amino acid sequence of hfT11 indicates that it corresponds to the C-terminal region of the PEVK segment of titin. The modular motifs of the open reading frame were revealed by protein analysis tools in MacVector and MACAW for homology and repeats. The sequence was divided to 28 sections, HRT1–28. The alignment of repeats (R, HRT1–4, HRT10–13, and HRT14–27) and two superrepeats (SRA, HRT14–20; SRB, HRT15–27) and nonrepeat (NR, HRT5–9) is indicated to the right. The consensus sequence for the PEVK modules is shown below. Prolines are ingreen, valines are in black, basic residues (Lys, Arg) are in blue, and acidic residues (Asp, Glu) are inred. The enclosed region, TP1 (468 residues,M r 51,491) was expressed in E. coli for the current study.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The PEVK region of human fetal skeletal muscle titin sequenced so far clearly indicates three levels of motifs: repeats of ∼28 residues, superrepeats of seven modules, and nonrepeat. The same research for internal repeats of human soleus PEVK (residues 0–2174, corresponding to 5618–7792 of EMBL X90569) revealed the same types of sequence motifs. The matrix plot in Fig. 2 B indicates significant repeats between residues 300 and 1840 of the PEVK segment of human soleus titin. Indeed, similar 28-residue repeats are abundant, and seven-module superrepeats are also present. The balance of the sequence is nonrepeat and contains clusters of acidic residues (see Refs. 2Freiburg A. Trombitas K. Hell W. Cazorla O. Fougerousse F. Centner T. Kolmerer B. Witt C. Beckmann J.S. Gregorio C.C. Granzier H. Labeit S. Circ. Res. 2000; 86: 1114-1121Crossref PubMed Scopus (272) Google Scholar and19Greaser M.L. Biophys. J. 2000; 78: 18AGoogle Scholar). In summary, PEVK of titin appears to be modular and consists mainly of tandem repeats of a fundamental 28-residue module interspersed in highly charged nonrepeat regions. The presence of the modular structure of PEVK, previously unrecognized, raised the question of whether PEVK may have stable structural folds. To explore this possibility, we expressed in Escherichia coli a 469-residue fragment (from 128–597, HRT5 to HRT21, designated as TP1) that includes both the nonrepeat and part of the superrepeat of PEVK. To avoid possible interference by expression tags in protein interaction studies, a nonfusion protein was produced. As shown in Fig. 3, TP1 was expressed as a soluble protein in reasonable yield. Due to its low UV absorption (a single tyrosine residue at position 155), purification and expression of TP1 were monitored by SDS gel electrophoresis and Western blotting with a monoclonal anti-titin, RT11. TP1 displayed unusually low SDS gel mobility and migrated with an apparent mass of 86 kDa. Moreover, TP1 was also difficult to transfer electrophoretically, and most TP1 stayed behind in the gel (Fig. 3 A, post-transfer gel pattern). The preferential transfer of smaller degradation products of TP1 gave a smear in Western blots that significantly underestimated the purity of TP1 (Fig. 3 A, RT11 Western blot). The low mobility may reflect either the lower than normal SDS binding or an extended conformation or stiffer SDS peptide complex for proline-rich peptides. For example, a proline-rich protein, calphotin (20Martin J.H. Benzer S. Rudnicka M. Miller C.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1531-1535Crossref PubMed Scopus (29) Google Scholar), displays a similar low motility. Purified TP1 is very soluble in a wide range of aqueous buffers and ionic strengths. The possible presence of secondary structure in TP1 was investigated by circular dichroism. CD spectra of TP1 display negative ellipticity with a minimum at 201 nm and a shoulder near 220 nm (Fig. 4 D). At first impression, these are characteristic spectra of polypeptides generally classified as random coils. Indeed, calculations for secondary structure content with several software programs indicate negligible α-helix and β-sheet structure (data not shown). However, further CD studies of TP1 subjected to urea and thermal titration raised doubts about this interpretation and led us to search for evidence for the presence of other stable structural features. Given the proline-rich content of TP1, we considered the possible presence of PPII. Such a left-handed helix, containing three trans-proline residues per turn, is found in polyproline in aqueous solution (21Bhatnagar R.S. Gough C.A. Fasman G.D. Circular Dichroism and Conformational Analysis of Biomolecules. Plenum Press, New York1996: 183-200Google Scholar, 25Sreerama N. Woody R.W. Biochemistry. 1994; 33: 10022-10025Crossref PubMed Scopus (261) Google Scholar) and is also present as short stretches in many globular proteins (22Adzhubei A.A. Sternberg M.J. J. Mol. Biol. 1993; 229: 472-493Crossref PubMed Scopus (428) Google Scholar). A detailed comparison of CD spectra of polyproline and TP1 strongly supports this notion. As shown in Fig. 4, A andB, CD spectra of polyproline show the characteristic strong negative band at 205 nm and a weak positive band at 229 nm of PPII helices (21Bhatnagar R.S. Gough C.A. Fasman G.D. Circular Dichroism and Conformational Analysis of Biomolecules. Plenum Press, New York1996: 183-200Google Scholar). Upon heating from 2 to 70 °C, both bands undergo incremental decrease in magnitude (Fig. 4 A,inset), indicating a loss of PPII helical content at higher temperature. This series of CD spectra (Fig. 4 A) displays an isodichroic point at 215 nm, indicating an equilibrium of two major populations of conformations. Another unique characteristic of PII is its response to urea and guanidinium chloride treatment (23Tiffany M.L. Krimm S. Biopolymers. 1973; 12: 575-587Crossref Scopus (122) Google Scholar). These chaotropic agents that commonly cause unfolding and loss of most secondary and tertiary structures in fact increase the helical content of PPII (23Tiffany M.L. Krimm S. Biopolymers. 1973; 12: 575-587Crossref Scopus (122) Google Scholar). As shown in Fig. 4 B, urea treatment up to 8m causes an increase in magnitude of bands at 205 and 229 nm, confirming an earlier report of the enhancement effect of urea on PPII content (23Tiffany M.L. Krimm S. Biopolymers. 1973; 12: 575-587Crossref Scopus (122) Google Scholar). The presence of an isodichroic point at 218 nm suggests again two populations of equilibrating conformations. High concentrations of urea progressively obscure the spectra below 205 nm and somewhat affect the accuracy of the isodichroic point (Fig.4 B, inset). The third characteristic behavior of PPII is the nearly linear response to thermal titration, without the sharp, sigmodial transition commonly observed for α-helices, β-sheets, and other stable folds that display cooperative melting. As shown in Fig. 4 C, thermal titration of polyproline from 2 to 90 °C, as monitored continuously at 201 nm, shows a liner response, with slight change in slopes at 28 and 80 °C. Such a titration curve suggests the gradual loss of PPII with raised temperature without cooperativity. The CD of TP1 and its responses to thermal and urea titration bear striking resemblance to those of polyproline under identical conditions (Figs. 4, D–F). The strong negative band at 200 nm is slightly blue-shifted from the 205-nm band of polyproline. The negative shoulder at 220 nm may be derived from the same transition as the 229-nm (positive) band of polyproline (Fig. 4, D andE). Upon heating from 2 to 70 °C, a significant decrease of the magnitude of both bands of TP1 occurs (Fig. 4 E,inset), showing the loss of stable folds. The presence of an isodichroic point near 210 nm between 2 and 50 °C suggests that at least two conformations are in equilibrium between below 50 °C (Fig.4 D). The addition of urea up to 8 m causes progressive decrease in the 200-nm band and an increase in the shoulder at 220 nm, with an isodichroic point at 210 nm (Fig. 4 E), consistent with the increase of PPII at higher concentration of urea. Thermal titration of TP1 at 201 nm reveals a linear response from 20 to
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