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• W1997036712 abstract "Integrin αIIbβ3clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of αIIbβ3. The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at ∼43 °C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen γ-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for αIIbβ3 complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 °C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for αIIbβ3 protomers and dimers based on the ectodomain structure of αvβ3. We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling. Integrin αIIbβ3clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of αIIbβ3. The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at ∼43 °C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen γ-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for αIIbβ3 complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 °C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for αIIbβ3 protomers and dimers based on the ectodomain structure of αvβ3. We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling. n-octyl-β-d-glucopyranoside cyclo(S,S)-l-lysyl-l-tyrosyl-glycyl-l-cystinyl-l-homoarginyl-glycyl-l-aspartyl-l-tryptophanyl-l-prolyl-l-cystine cyclo(S,S)-l-lysyl-l-tyrosyl-glycyl-l-cystinyl-l-arginyl-glycyl-l-aspartyl-l-tryptophanyl-l-prolyl-l-cystine weight average molecular weight Integrins are a widely distributed family of heterodimeric transmembrane receptors that anchor cells to extracellular matrix proteins and mediate two-way communication between the exterior and interior of a cell (1Critchley D.R. Holt M.R. Barry S.T. Priddle H. Hemmings L. Norman J. Biochem. Soc. Symp. 1999; 65: 79-99Google Scholar, 2Hynes R.O. Cell. 1992; 69: 11-25Google Scholar). The αIIbβ3complex is the classic example of a regulated integrin, a receptor whose affinity for adhesive macromolecules is modulated by changes in conformation and clustering (3Shattil S.J. Thromb. Haemostasis. 1999; 82: 318-325Google Scholar, 4Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Google Scholar, 5Liddington R.C. Bankston L.A. Exp. Cell Res. 2000; 261: 37-43Google Scholar). The receptor is maintained in a default inactive state on circulating human blood platelets, possibly by interactions between the short cytoplasmic domains of the αIIb and β3 subunits or with integrin-associated proteins (6Vinogradova O. Haas T. Plow E.F. Qin J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1450-1455Google Scholar, 7Ginsberg M.H. O'Toole T.E. Loftus J.C. Plow E.F. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 221-231Google Scholar, 8Brown E.J. Frazier W.A. Trends Cell Biol. 2001; 11: 130-135Google Scholar, 9Weljie A.M. Hwang P.M. Vogel H.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 30: 5878-5883Google Scholar). According to this model, proteins like fibrinogen or fibronectin are prevented from binding to extracellular region of αIIbβ3 until a platelet stimulus, such as thrombin, binds to a G-protein-coupled receptor (3Shattil S.J. Thromb. Haemostasis. 1999; 82: 318-325Google Scholar, 10Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Google Scholar, 11Payrastre B. Missy K. Trumel C. Bodin S. Plantavid M. Chap H. Biochem. Pharmacol. 2000; 60: 1069-1074Google Scholar). A rapid cascade of intracellular events releases an inhibitory lock, sending a signal some 15 nm outwards to the ectodomain of αIIbβ3, where a rearrangement of intersubunit contacts leads to an “open” receptor with a functional binding site (12Faull R.J. Ginsberg M.H. J. Am. Soc. Nephrol. 1996; 7: 1091-1097Google Scholar, 13Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar, 14Sims P.J. Ginsberg M.H. Plow E.F. Shattil S.J. J. Biol. Chem. 1991; 266: 7345-7352Google Scholar). This process of converting αIIbβ3 to an active form is referred to as “inside-out” signaling (3Shattil S.J. Thromb. Haemostasis. 1999; 82: 318-325Google Scholar). Receptor occupancy then sends an “outside-in” signal, leading to integrin clustering and downstream activation of kinases, especially focal adhesion kinase, that stabilize the integrin-mediated links between extracellular matrix proteins and actin filaments (15Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Google Scholar, 16Hartwig J.H. Barkalow K. Azim A. Italiano J. Thromb. Haemostasis. 1999; 82: 392-398Google Scholar, 17Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2000; 275: 22607-22610Google Scholar). Clustering of bound receptors may facilitate these processes by increasing the local concentration of integrin-associated proteins, especially those that bind to the cytoplasmic tail of β3(4Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Google Scholar, 18Hato T. Pampori N. Shattil S.J. J. Cell Biol. 1998; 141: 1685-1695Google Scholar, 19Liu S. Calderwood D.A. Ginsberg M.H. J. Cell Sci. 2000; 113: 3563-3571Google Scholar). A positive feedback mechanism may also be at work on the cell surface, in that integrin oligomers could be especially efficient at capturing multimeric adhesive proteins (15Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Google Scholar, 20Miyamoto S. Akiyama S.K. Yamada K.M. Science. 1995; 267: 883-885Google Scholar). Our understanding of the molecular basis for integrin activation has been considerably enhanced by the recent publication of crystal structures for the extracellular domain of the αvβ3 integrin, in the absence and presence of ligand (21Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Google Scholar, 22Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Google Scholar). However, many questions remain about the mechanistic details of the biomechanical coupling between the distant intra- and extracellular domains of αIIbβ3. Previous experiments have shown that truncating either the αIIb or β3 cytoplasmic regions yields a receptor that is constitutively active for inside-out signaling, indicating that communication between the cytoplasmic domains maintains the inactive state of the integrin (23O'Toole T.E. Mandelman D. Forsyth J. Shattil S.J. Plow E.F. Ginsberg M.H. Science. 1991; 254: 845-847Google Scholar, 24O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Google Scholar). We have recently shown that a truncation mutant, lacking the αIIb cytoplasmic domain, underwent further activation in the presence of a high affinity fibrinogen mimetic peptide resulting in the formation of oligomers (25Hantgan R.R. Stahle M. Del G.V. Adams M. Lasher T. Jerome W.G. McKenzie M. Lyles D.S. Biochim. Biophys. Acta. 2001; 1540: 82-95Google Scholar). These observations reinforce the concept that clustering plays a major role in the regulation of integrin affinity through outside-in signaling (13Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar, 15Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Google Scholar, 26Li R. Babu C.R. Lear J.D. Wand A.J. Bennett J.S. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12462-12467Google Scholar). Concerning the forces that drive integrin clustering, we and others have demonstrated that the ability of RGDX peptides to block the function of αIIbβ3 (27Tranqui L. Andrieux A. Hudry-Clergeon G. Ryckewaert J.J. Soyez S. Chapel A. Ginsberg M.H. Plow E.F. Marguerie G. J. Cell Biol. 1989; 108: 2519-2527Google Scholar) as well as to perturb the conformation of its ectodomain and to promote “tail to tail” oligomerization all increased with the hydrophobicity of the residue in the X-position (28Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14464Google Scholar, 29Hantgan R.R. Stahle M.C. Jerome W.G. Nagaswami C. Weisel J.W. Thromb. Haemostasis. 2002; 87: 910-917Google Scholar). Likewise, we found that eptifibatide, a cyclized fibrinogen-mimetic peptide with a tryptophan residue in its integrin-targeting sequence (30Scarborough R.M. Naughton M.A. Teng W. Rose J.W. Phillips D.R. Nannizzi L. Arfsten A. Campbell A.M. Charo I.F. J. Biol. Chem. 1993; 268: 1066-1073Google Scholar), was especially effective at promoting αIIbβ3oligomerization, as monitored by sedimentation equilibrium and electron microscopy (31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar). These observations have led to the concept that nonpolar receptor-ligand interactions contribute to both integrin activation and self-association (28Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14464Google Scholar, 31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar). This article tests that hypothesis by investigating the effects of both temperature and ligand binding on αIIbβ3structure/stability. If hydrophobic effects are important in these interactions, thermodynamic principles (32Klotz I.M. Darnall D.W.L.N.R. Neurath H. Hill R.L. The Proteins. Academic Press, New York1975: 293-411Google Scholar, 33Van Holde K.E. Johnson W.C.H.P.S. Macromolecules in Solution: Thermodynamics and Equilibria, Principles of Physical Biochemistry. Prentice Hall, Upper Saddle River, NJ1998Google Scholar, 34Calderone C.T. Williams D.H. J. Am. Chem. Soc. 2001; 123: 6262-6267Google Scholar) and experience with other self-assembling systems (35Pandit A. Visschers R.W. van Stoleleum I.H.M. Kraayenhof R. van Grondelle R. Biochemistry. 2001; 40: 12913-12924Google Scholar, 36Clantin B. Tricot C. Lonhienne T. Stalon V. Villeret V. Eur. J. Biochem. 2001; 268: 3937-3942Google Scholar, 37Fieber W. Schneider M.L. Matt T. Krautler B. Konrat R. Bister K. J. Mol. Biol. 2001; 307: 1395-1410Google Scholar) predict that the extent of αIIbβ3 oligomerization should increase with increasing temperature. However, extracting thermodynamic data on a transmembrane protein such as the αIIbβ3integrin requires pure protein isolated in a thermally stable environment amenable to spectroscopic studies. The neutral, nondenaturing detergent octyl glucoside (OG)1 (38Garavito R.M. Ferguson-Miller S. J. Biol. Chem. 2001; 276: 32403-32406Google Scholar, 39Bogusz S. Venable R.M. Pastor R.W. J. Phys. Chem. 2000; 104: 5462-5470Google Scholar) is especially well suited for biophysical characterizations of integral membrane proteins because it exhibits a critical micellar concentration and aggregation number that are nearly invariant from 20 to 60 °C (40Aoudia M. Zana R. J. Colloid Interface Sci. 1998; 206: 158-167Google Scholar). Octyl glucoside has been used to study the structure and dynamics of bacteriorhodopsin (41Gottschalk M. Dencher N.A. Halle B. J. Mol. Biol. 2001; 311: 605-621Google Scholar), mammalian rhodopsin (42Klein-Seetharaman J. Reeves P.J. Loewen M.C. Getmanova E.V. Chung J. Schwalbe H. Wright P.E. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3452-3457Google Scholar), OmpF porin fromEscherichia coli (43Pebay-Peyroula E. Garavito R.M. Rosenbusch J.P. Zulauf M. Timmins P.A. Structure. 1995; 3: 1051-1059Google Scholar), and the E. coli outer membrane ferrichrome transporter FhuA (44Boulanger P. le Maire M. Bonhivers M. Dubois S. Desmadril M. Letellier L. Biochemistry. 1996; 35: 14216-14224Google Scholar). Extensive biophysical characterizations have shown that octyl glucoside, even at a concentration well above its critical micellar concentration, does not perturb the quaternary structure of the soluble lens protein α-crystallin (45Aerts T. Clauwaert J. Haezebrouck P. Peeters E. Van Dael H. Eur. Biophys. J. 1997; 25: 445-454Google Scholar, 46Vanhoudt J. Abgar S. Aerts T. Clauwaert J. Biochemistry. 2000; 39: 4483-4492Google Scholar). Furthermore, oligomerization studies of subunits B777 (47Gall A. Dellerue S. Lapouge K. Robert B. Bellissent-Funel M.C. Biopolymers. 2001; 58: 231-234Google Scholar) and B820 (35Pandit A. Visschers R.W. van Stoleleum I.H.M. Kraayenhof R. van Grondelle R. Biochemistry. 2001; 40: 12913-12924Google Scholar) of the light-harvesting complex from Rhodobacter sphearoides have been performed in octyl glucoside micelles. Based on our experience (28Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14464Google Scholar, 31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar, 49Hantgan R.R. Braaten J.V. Rocco M. Biochemistry. 1993; 32: 3935-3941Google Scholar) and that of others (50Makogonenko E.M. Yakubenko V.P. Ingham K.C. Medved L.V. Eur. J. Biochem. 1996; 237: 205-211Google Scholar) with biophysical characterizations of the αIIbβ3integrin in octyl glucoside, we recognize the limitations of extrapolation from a detergent-solubilized protein to the physiological situation. However, the responses of solubilized integrins to ligand binding, such as conformational changes and oligomerization (28Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14464Google Scholar, 31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar), have been remarkably similar to the effects seen in cellular membranes (20Miyamoto S. Akiyama S.K. Yamada K.M. Science. 1995; 267: 883-885Google Scholar, 51Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Google Scholar). In addition, Litvinov et al. (52Litvinov R.I. Shuman H. Bennett J.S. Weisel J.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7426-7431Google Scholar) recently demonstrated that both purified αIIbβ3 and αIIbβ3 on the platelet surface displayed comparable force histograms for fibrinogen binding. These observations probably reflect the fact that the ectodomain of αIIbβ3 contains >90% of the integrin residues and only one narrow nonpolar segment on each subunit traverses the plasma membrane (28Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14464Google Scholar). Thus the model of a circumferential transmembrane belt comprised of a limited number of protein-bound detergent (43Pebay-Peyroula E. Garavito R.M. Rosenbusch J.P. Zulauf M. Timmins P.A. Structure. 1995; 3: 1051-1059Google Scholar, 53Roth M. Bentley-Lewit A. Michel H. Deisenhofer J. Huber R. Oesterhelt D. Nature. 1989; 340: 659-662Google Scholar) or lipid molecules (54Luecke H. Schobert B. Richter H.T. Cartailler J.P. Lanyi J.K. J. Mol. Biol. 1999; 291: 899-911Google Scholar, 55Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Google Scholar) established from high resolution crystal structures of integral membrane proteins supports the validity of the studies of αIIbβ3 in octyl glucoside micelles described here. Cyclo(S,S)-l-lysyl-l-tyrosyl-glycyl-l-cystinyl-l-homoarginyl-glycyl-l-aspartyl-l-tryptophanyl-l-prolyl-l-cystine (cHArGD) and cyclo(S,S)-l-lysyl-l-tyrosyl-glycyl-l-cystinyl-l-arginyl-glycyl-l-aspartyl-l-tryptophanyl-l-prolyl-l-cystine (cRGD) (31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar) were synthesized and purified by the Protein Analysis Core Laboratory of the Comprehensive Cancer Center of Wake Forest University (Winston-Salem, NC) using previously described protocols (56Hantgan R.R. Endenburg S.C. Cavero I. Marguerie G. Uzan A. Sixma J.J. De Groot P.G. Thromb. Haemostasis. 1992; 68: 694-700Google Scholar). Each peptide was shown to have the correct amino acid sequence and the correct molecular mass, using an Applied Biosystems 475 automated peptide synthesizer and a Quattro II triple quadropole mass spectrometer (Micromass, Inc., Beverly, MA), respectively. Eptifibatide, (N 6-(aminoiminomethyl)-N 2-(3-mercapto-1-oxopropyl-l-lysylglycyl-l-α-aspartyl-l-tryptophanyl-l-prolyl-cysteinamide, cyclic (1Critchley D.R. Holt M.R. Barry S.T. Priddle H. Hemmings L. Norman J. Biochem. Soc. Symp. 1999; 65: 79-99Google Scholar, 2Hynes R.O. Cell. 1992; 69: 11-25Google Scholar, 3Shattil S.J. Thromb. Haemostasis. 1999; 82: 318-325Google Scholar, 4Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Google Scholar, 5Liddington R.C. Bankston L.A. Exp. Cell Res. 2000; 261: 37-43Google Scholar, 6Vinogradova O. Haas T. Plow E.F. Qin J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1450-1455Google Scholar)-disulfide), was kindly provided by COR Therapeutics (San Francisco, CA) where it has been developed as a pharmaceutical, Integrilin (57Phillips D.R. Scarborough R.M. Am. J. Cardiol. 1997; 80: 11B-20BGoogle Scholar). Peptide concentrations were determined by quantitative amino acid composition analyses as previously described (31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar, 56Hantgan R.R. Endenburg S.C. Cavero I. Marguerie G. Uzan A. Sixma J.J. De Groot P.G. Thromb. Haemostasis. 1992; 68: 694-700Google Scholar). Highly purified human fibrinogen (free of plasminogen and Factor XIII) was purchased from American Diagnostica (Greenwich, CT) and highly purified human α-thrombin was from Sigma. Platelet-rich plasma and gel-filtered platelets were isolated from blood obtained by venipuncture from healthy, adult, volunteer donors as previously described (58Hantgan R.R. Blood. 1984; 64: 896-906Google Scholar). Platelet counts were determined with a Coulter MDII Cell Counter (Beckman Instruments, Miami, FL). Platelet αIIbβ3 occupancy was determined with flow cytometric analysis using a Biocytex kit BX7001 (Marseilles, France) (25Hantgan R.R. Stahle M. Del G.V. Adams M. Lasher T. Jerome W.G. McKenzie M. Lyles D.S. Biochim. Biophys. Acta. 2001; 1540: 82-95Google Scholar, 59Quinn M. Deering A. Stewart M. Cox D. Foley B. Fitzgerald D. Circulation. 1999; 99: 2231-2238Google Scholar). Platelet aggregation profiles were obtained in a Chrono-Log model 500 aggregometer. Platelet adhesion to fibrin was determined in a microtiter plate adhesion assay with colorimetric read-out as previously described (25Hantgan R.R. Stahle M. Del G.V. Adams M. Lasher T. Jerome W.G. McKenzie M. Lyles D.S. Biochim. Biophys. Acta. 2001; 1540: 82-95Google Scholar). Milligram quantities of highly purified αIIbβ3 were isolated from outdated human blood platelets (American Red Cross, Triad Blood Center, Winston-Salem, NC) as previously described (28Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14464Google Scholar, 49Hantgan R.R. Braaten J.V. Rocco M. Biochemistry. 1993; 32: 3935-3941Google Scholar). Biophysical measurements were performed on peak integrin fractions obtained by size exclusion chromatography at 4 °C on a 0.9 × 85-cm column of Sephacryl S-300 equilibrated in pH 7.4 buffer (HSC-OG) containing 0.13 mol/liter NaCl, 0.01 mol/liter HEPES, 0.002 mol/liter CaCl2, 3 × 10−8 mol/liter basic trypsin inhibitor, 10−6 mol/liter leupeptin, 0.02% sodium azide, and 0.03 mol/litern-octyl-β-d-glucopyranoside. Peak fractions were then concentrated in an Amicon pressure concentrator with a PLHK cellulose membrane, 100,000 Da retention limit (31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar). UV absorbance measurements were performed as a function of wavelength on a Beckman diode array spectrophotometer with αIIbβ3 samples contained in 1-cm path length, 0.1-ml volume quartz cuvettes. Circular dichroic spectroscopy was performed as a function of temperature from 20 to 60 °C in a Jasco model 720 spectropolarimeter (Japan Spectroscopic Co., Tokyo) with samples in a thermostatted 0.05-cm path-length cuvette; data are expressed as molar ellipticity, [Θ],versus wavelength (25Hantgan R.R. Stahle M. Del G.V. Adams M. Lasher T. Jerome W.G. McKenzie M. Lyles D.S. Biochim. Biophys. Acta. 2001; 1540: 82-95Google Scholar). In computing [Θ], each data set was corrected for the signal from the HSC-OG buffer or buffered octyl glucoside containing the ligand-mimetic peptide cHArGD at the same temperature. However, these background measurements changed by only ±5% over the range 20–60 °C. Fluorescence emission spectra were obtained over the same temperature range with an Aminco-Bowman series 2 luminescence spectrometer (SLM-Aminco, Rochester, NY) with samples contained in thermostatted quartz microcuvettes (Hellma Cells, Inc., White Plains, NY); an excitation wavelength at 278 nm was used (31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar). These data were also corrected for background fluorescence; the signal from HSC-OG buffer was found to vary by ±6% over the range 20–60 °C. The small fluorescence intensity from the tryptophan-containing ligand-mimetic peptide cHArGD in HSC-OG exhibited an ∼2-fold hyperbolic decrease with increasing temperature, as expected for intrinsic fluorescence measurements (60Cantor C.R. Schimmel P.R. Biophysical Chemistry. Part II: Techniques for the Study of Biological Structure and Function. W. H. Freeman and Co., San Francisco1980Google Scholar). Static and dynamic light scattering measurements were performed in a Brookhaven Instruments BI-2030 AT correlator operated in conjunction with a BI-200 SM light scattering photometer/photon counting detector and a Spectra Physics 127 He-Ne laser (28Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14464Google Scholar); samples in quartz microcuvettes were contained in a thermostatted refractive index matching vat at temperatures in the range 20–40 °C. For translational diffusion coefficient determinations, each intensity-normalized photon count autocorrelation function obtained for the αIIbβ3 complex was first corrected for the contributions of octyl glucoside micelles. Following procedures described in our earlier work (49Hantgan R.R. Braaten J.V. Rocco M. Biochemistry. 1993; 32: 3935-3941Google Scholar), the autocorrelation function was treated as an intensity-weighted sum of two exponential decay components, one corresponding to the macromolecule and the other to the faster moving detergent micelles. The contribution of the detergent was determined from separate measurements of buffered octyl glucoside, obtained under the same instrumental conditions. The resultant signal was weighted by its fractional intensity, then subtracted from the corresponding autocorrelation function for the integrin:detergent mixture to yield a corrected autocorrelation function. Each corrected autocorrelation function was then analyzed by the method of cumulants to obtain a z-average translational diffusion coefficient for the αIIbβ3 complex. Size distribution information was also obtained from these corrected autocorrelation functions with the CONTIN algorithm (61Provencher S.W. Comp. Phys. Commun. 1982; 27: 229-242Google Scholar). We note that CONTIN analysis did not consistently identify a peak corresponding to the ∼5-nm diameter OG micelles in the uncorrected data, probably because we worked under conditions where the detergent contributed less than 20% of the total scattering intensity. All translational diffusion coefficients reported here have been corrected for solvent viscosity to obtain D 20,w and Stokes radius (R s) values. Data collected from buffered HSC-OG indicated that the octyl glucoside micelle size distribution did not change appreciably from 20 to 40 °C in that the scattering intensity varied by ±13% and the mean particle diameter changed by ±12%; no temperature-dependent trends were observed in these data. Our results obtained with 30 mm OG are consistent with the report of Aoudia and Zana (40Aoudia M. Zana R. J. Colloid Interface Sci. 1998; 206: 158-167Google Scholar), who found that both critical micellar concentration for octyl glucoside (23–25 mm) and aggregation number (107-92) exhibited minimal temperature dependence from 20 to 60 °C. Sedimentation velocity and equilibrium measurements were performed in a Beckman Optima XL-A analytical ultracentrifuge (Beckman) equipped with absorbance optics and an An60 Ti rotor (28Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14464Google Scholar, 31Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Google Scholar). By always including HSC-OG buffer or buffer + ligand-mimetic peptide in the reference compartment, the resultant optical signals were corrected for any temperature-induced effects on the solvent, although data previously described demonstrated these effects to be minimal. Sedimentation velocity data obtained at 20 and 40 °C were analyzed using both SVEDBERG (version 1.04) and DCDT+ (version 6.31) software (J. Philo, Thousand Oaks, CA) to obtain the weight average sedimentation coefficient (s w) and distribution of sedimenting species, g(s*), respectively (62Stafford III, W.F. Anal. Biochem. 1992; 203: 295-301Google Scholar). All sedimentation coefficients reported here have been corrected for solvent density and viscosity to obtains 20,w values. The absorbance versus radial distance data obtained by sedimentation equilibrium were analyzed by nonlinear regression with WinNONLIN3 (63Johnson M. Correia J.J. Yphantis D.A. Halvorson H. Biophys. J. 1981; 36: 575-588Google Scholar) to obtain weight average molecular weights (M w) for the αIIbβ3complex alone and in the presence of ligand-mimetic peptide.M w data were analyzed for their dependence on rotor speed (at 6000 and 8000 rpm), a diagnostic for irreversible aggregation (64Laue T.M. Methods Enzymol. 1995; 259: 427-452Google Scholar). In the absence of ligands, data obtained with αIIbβ3 yielded a ratioM w (6000 rpm)/M w (8000 rpm) of 0.905 at 30 °C, 0.839 at 37 °C, and 0.737 at 40 °C. In addition, global fits to data obtained at 37 and 40 °C yielded a variance of ∼3 × 10−3, a 5-fold decrease in quality compared with fits obtained with data at lower temperatures. These observations are indicative of thermal aggregation for the ligand-free integrin (64Laue T.M. Methods Enzymol. 1995; 259: 427-452Google Scholar). Because depletion of larger species can also influence the resultantM w data, the quantity of absorbing material present in cells containing αIIbβ3 at equilibrium was computed and compared with that initially added. This was done by numerical integration of the absorbance versusradial distance profiles obtained at 30 and 40 °C, as well as the initial, pre-equilibrium scans obtained at 3000 rpm. Whereas ∼100% recovery was achieved at 6000 rpm/30 °C, this parameter fell to ∼68% at 8000 rpm/30 °C; because only absorbance values <3.0 were included, there is some uncertainty associated with these estimates. In addition, only data at absorbence <1.5 were included in the subsequent analyses to ensure compliance with Beer's law, thus 47% of the integrated area was used for fitting at 6000 rpm and 42% at 8000 rpm. These effects became more pronounced at 40 °C where recoveries of 42 and 28% were obtained at 6000 and 8000 rpm, respectively; 33% of the 6000 rpm data and 25% of the 8000 rpm data were used for fitting. In contrast, M w data obtained for the ligand-bound integrins showed a somewhat less pronounced dependence on rotor speed and temperature: for example, the ratioM w (6000 rpm)/M w (8000 rpm) was 0.907 at 30 °C, 0.851 at 37 °C, and 0.765 at 40 °C for the αIIbβ3·cHArGD complex. Global fits of data obtained in this temperature range, at two rotor speeds, and receptor concentrations in the range 1.4 to 4.0 μm, yielded a variance of ∼7 × 10−4, indicating the data obtained for the ligand-bound integrin were more consistent with oligomerization, rather than the aggregation behavior observed for the free receptor. Sample depletion was also less pronounced with the ligand-bound integrin samples, as recoveries of 94 ± 10% and fitted ranges of 42 ±" @default.
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• W1997036712 date "2003-01-01" @default.
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• W1997036712 title "Ligand Binding Promotes the Entropy-driven Oligomerization of Integrin αIIbβ3" @default.
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• W1997036712 doi "https://doi.org/10.1074/jbc.m208869200" @default.
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