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• W2038952850 abstract "α-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-α (Gro-α) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-α give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, 15N/1H heteronuclear single quantum coherence NMR data demonstrate loss of 1H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-α in particular, 1H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein. α-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-α (Gro-α) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-α give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, 15N/1H heteronuclear single quantum coherence NMR data demonstrate loss of 1H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-α in particular, 1H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein. fibroblast growth factor nuclear Overhauser effect two-dimensional NMR nuclear Overhauser effect spectroscopy heteronuclear single quantum coherence three-dimensional1H-13C-13C-1H total correlated spectroscopy glycosaminoglycan platelet factor-4 N-terminal chimera of PF4 interleukin-8 growth-related protein-α high performance liquid chromatography heparin dodecasaccharide heparin-induced thrombocytopenia/thrombosis Heparin is a polydisperse, sulfated copolymer of 1–4-linked glucosamine and uronic acid residues. Most of the heparin molecule is accounted for by this repeating disaccharide unit, which consists primarily of 2-O-sulfated-α-l-idopyranosyluronic acid 2-sulfate and 2-deoxy-2-sulfamido-α-d-glucopyranose (GlcNSO3) 6-sulfate (1Jeanloz R.W. Adv. Exp. Med. Biol. 1975; 52: 3-12Crossref PubMed Scopus (13) Google Scholar). Fig. 1 illustrates the covalent structure of a heparin dodecasaccharide. A number of proteins have been identified that bind avidly to polyanionic heparin and heparin-derived polysaccharides, e.g. fibroblast growth factor-2 (FGF-2)1 (2Tyrrell D.J. Ishihara M. Rao N. Horne A. Kiefer M.C. Stauber G.B. Lam L.H. Stack R.J. J. Biol. Chem. 1993; 268: 4684-4689Abstract Full Text PDF PubMed Google Scholar), hepatocyte growth factor (3Lyon M. Deakin J.A. Mizuno K. Nakamura T. Gallagher J.T. J. Biol. Chem. 1994; 269: 11216-11223Abstract Full Text PDF PubMed Google Scholar), antithrombin-III (4von Boeckel C.A.A. Grootenhuis P.D.J. Visser A. Struct. Biol. 1994; 1: 423-425Crossref PubMed Scopus (93) Google Scholar), and α-chemokines (5Mire-Slius, A. & Thorpe, R. (eds) (1998) Cytokines, Academic Press, San DiegoGoogle Scholar) like platelet factor-4 (PF4) and growth-related protein-α (Gro-α). α-Chemokines, which demonstrate a variety of physiological activities, some of which may be related to binding heparin-like glycosaminoglycans on the surface of cells, present good model systems for exploring heparin-protein interactions. PF4 is perhaps the strongest heparin-binding protein known (6Witt D.P. Lander A.D. Curr. Biol. 1994; 4: 394-400Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). During coagulation, for example, PF4 displaces thrombin from heparin, where it forms anticoagulant complex with antithrombin-III, into solution, where thrombin is procoagulant through its interactions with blood clotting factors (7Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1624) Google Scholar). PF4 exhibits other physiological effects (5Mire-Slius, A. & Thorpe, R. (eds) (1998) Cytokines, Academic Press, San DiegoGoogle Scholar), including stimulation of fibroblast attachment to the substrate, chemotactic activity with respect to neutrophils, monocytes and fibroblasts, potentiation of platelet aggregation and inhibition of megakaryocytopoiesis, angiogenesis, and solid tumor growth. Gro-α (also called melanoma growth stimulatory activity (MGSA)) is secreted by a number of different cells and demonstrates a variety of biological activities (5Mire-Slius, A. & Thorpe, R. (eds) (1998) Cytokines, Academic Press, San DiegoGoogle Scholar), including stimulation of melanoma cell growth, neutrophil chemotaxis, and inhibition of collagen expression. α-Chemokines can exist as dimers or tetramers (5Mire-Slius, A. & Thorpe, R. (eds) (1998) Cytokines, Academic Press, San DiegoGoogle Scholar). In solution, PF4 may form dimers and tetramers, whereas Gro-α and interleukin-8 (IL-8) are observed to aggregate only to the dimer state. Structure analysis of native human PF4 (8Zhang X. Chen L. Bancroft D.P. Lai C., K. Maione T.E. Biochemistry. 1994; 33: 8361-8366Crossref PubMed Scopus (158) Google Scholar) indicates that each subunit (A, B, C, and D) of the tetramer has a three-stranded β-sheet scaffold onto which is folded an amphipathic C-terminal α-helix and an aperiodic N-terminal segment. In general, such tetrameric structures naturally have three types of monomer-monomer (dimer) interactions, which are referred to in the literature as AB, AC and AD. In AB-type dimers, monomers associate by continuing their anti-parallel β-sheets to form a six stranded sheet. Gro-α and IL-8 associate as AB-type dimers. The general AB-type dimer fold that is observed in all α-chemokines is illustrated in Fig. 2 for PF4 and Gro-α. In the crystal (8Zhang X. Chen L. Bancroft D.P. Lai C., K. Maione T.E. Biochemistry. 1994; 33: 8361-8366Crossref PubMed Scopus (158) Google Scholar), as well as in solution (9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar), native PF4 AB-type dimers associate to form tetramers that are asymmetric, i.e. each monomer in the tetramer has a slightly different orientation with respect to any other monomer subunit. In NMR spectra, this gives rise to multiple sets of resonances for each residue, one set from each subunit, making NMR solution studies with native PF4 intractable. Substitution of a highly acidic N-terminal region of PF4 with the N-terminal sequence of homologous IL-8 results in formation of a symmetric tetramer (9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar) wherein each monomer is identically positioned within the tetramer, thus facilitating interpretation of NMR experiments. This chimera, named PF4-M2, binds heparin nearly as tightly as native PF4 (9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar) and has been used in the present heparin binding studies. The C terminus of PF4 (… LY60KKIIK65KLLES70) (and of α-chemokines in general) was proposed originally to be the primary mediator of heparin binding (10Loscalzo J. Melnick B. Handin R.I. Arch. Biochem. Biophys. 1985; 240: 446-455Crossref PubMed Scopus (103) Google Scholar, 11Cowan S.W. Bakshi E.N. Machin K.J. Isaacs N.W. Biochem. J. 1986; 234: 485-488Crossref PubMed Scopus (27) Google Scholar), particularly via interactions with the four lysine residues conformed spatially in tandem on the solvent-exposed surface of the C-terminal α-helix. In fact, a single amino acid replacement of Lys-65 (PF4) by Glu-63 (Gro-α) (… IVKKIIE63KMLNSDKS) was used to rationalize heparin binding differences between these two proteins (6Witt D.P. Lander A.D. Curr. Biol. 1994; 4: 394-400Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Analysis of the PF4 tetrameric structure, however, reveals the presence of a ring of positive charge circumventing the molecule and running perpendicular to the C-terminal helices (8Zhang X. Chen L. Bancroft D.P. Lai C., K. Maione T.E. Biochemistry. 1994; 33: 8361-8366Crossref PubMed Scopus (158) Google Scholar, 9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar). No experimentally determined structure of the PF4-heparin complex is yet known. However, two models for heparin binding to PF4 have been devised in which the heparin molecule binds either parallel (11Cowan S.W. Bakshi E.N. Machin K.J. Isaacs N.W. Biochem. J. 1986; 234: 485-488Crossref PubMed Scopus (27) Google Scholar) or perpendicular (12Stuckey J.A. St. Charles R. Edwards B.F.P. Proteins Struct. Funct. Genet. 1992; 14: 277-287Crossref PubMed Scopus (89) Google Scholar) to the C-terminal α-helixes of the AB dimer (see Fig. 2). In an experimental NMR study, Mayo et al. (13Mayo K.H. Roongta V. Ilyina E. Dundas M. Joseph J. Lai C.K. Maione T. Daly T.J. Biochem. J. 1995; 312: 357-365Crossref PubMed Scopus (100) Google Scholar) deduced that the perpendicular binding mode is most probably favored and that, even though heparin (9,000-dalton cut-off) interacted with residues within the ring of positively charged groups, Arg-20 and Arg-22 were crucial to the binding process, more so than the C-terminal lysines. These and most other protein-heparin binding studies have been performed using commercial preparations of heparin that result in limited structural information due to heterogeneity of the heparin polymer. Moreover, these heparin preparations are known to induce protein precipitation when the heparin:protein ratio is less than about 3:1. One way to avoid these problems is to use heparinase-derived, homogeneous preparations of short-chain heparins. Heparin chains up to tetradecasaccharide in length can be obtained (14Pervin A. Gallo C. Jandik K. Han X.-J. Linhardt R.J. Glycobiology. 1995; 5: 83-95Crossref PubMed Scopus (195) Google Scholar); however, for milligram quantities required for NMR studies, a dodecasaccharide is probably the best attainable. The present study, therefore, is aimed at investigating interactions of a heparin-derived dodecasaccharide (H12) (Fig. 1) with PF4-M2 and Gro-α. Approximately 2 g of heparin was dissolved in 100 ml of distilled water and dialyzed exhaustively, freeze-dried, and prepared at exactly 20 mg/ml in distilled water (15Linhardt R.J. Rice K.G. Kim Y.S. Lohse D.L. Wang H.M. Loganathan D. Biochem. J. 1988; 254: 781-787Crossref PubMed Scopus (128) Google Scholar). To 50 ml of heparin (20 mg/ml) was added 450 ml of sodium phosphate buffer (55 mm sodium phosphate, pH 7) containing 800 mIU of heparinase. The reaction mixture was incubated at 30 °C for 20 h. At 30% reaction completion, reaction was terminated by heating to 100 °C. The depolymerization mixture was separated, and individual low molecular weight heparin oligosaccharide fractions were isolated by gel permeation chromatography and strong-anion exchange HPLC as discussed previously (14Pervin A. Gallo C. Jandik K. Han X.-J. Linhardt R.J. Glycobiology. 1995; 5: 83-95Crossref PubMed Scopus (195) Google Scholar). The heparin oligosaccharide used in the present study is a dodecasaccharide with the structure shown in Fig. 1. In the present report, this heparin-derived dodecasaccharide is referred to as H12. This procedure produced milligram quantities of H12 necessary for the NMR studies; nonetheless, amounts of H12 were limited to about 4 mg. Synthetic genes for PF4-M2 and Gro-α were expressed as non-fusion proteins in Escherichia coli BL21 cells and grown at the 10-liter scale with 15N-enriched ammonia as the nitrogen source and [13C]glucose as the carbon source (9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar). Proteins were purified and refolded essentially as described previously (16Yang Y. Mayo K.H. Daly T.J. Barry J.K. La Rosa G.L. J. Biol. Chem. 1994; 269: 20110-20118Abstract Full Text PDF PubMed Google Scholar). Purity was assessed by Coomassie staining of SDS-polyacrylamide gel electrophoresis, analytical C4 reverse phase HPLC, and amino acid analysis. Typically, several hundred milligrams of greater than 95% pure material was isolated from 100 g of starting material. Protein concentration was determined by using the bicinchoninic acid (BCA) assay (17Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18590) Google Scholar) and calculated on the basis of protein concentrations obtained from a standard dilution series of bovine serum albumin. Protein concentrations also were checked by using the methods of Lowry et al. (18Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-270Abstract Full Text PDF PubMed Google Scholar) and of Waddell (19Waddell W.J. J. Lab. Clin. Med. 1956; 48: 311-314PubMed Google Scholar). 15N/13C-labeled PF4-M2 was dissolved in 10%/90% D2O/H2O at a concentration of 6 mg/ml, pH 5.9, 20 mm NaCl. This PF4-M2 solution was then titrated into a solution of H12 in aliquots of 30 μl to reach the following PF4-M2 (monomeric):H12 ratios: 1:6, 1:3, 1:2, 1:1, 2:1, and 4:1. To achieve the final ratio, unlabeled PF4-M2 was added to the 2:1 mixture of 15N/13C-labeled PF4-M2 and H12. The addition of each aliquot of PF4-M2 solution induced some opacity to the H12 solution, which became clear after heating up to 50 °C for about 30 min or sitting overnight at room temperature. Following mixing of H12 and PF4-M2, the solution pH remained essentially unchanged, varying by no more than 0.05 pH units. 15N-Labeled Gro-α was dissolved in 10%/90% D2O/H2O at concentration of 10 mg/ml. In order to facilitate NMR assignments, the solution pH was adjusted to 5.7 to be the same as that used in the assignment/structural studies of Fairbrother et al. (20Fairbrother W.J. Reilly D. Colby T. Hesselgesser J. Horuk R. J. Mol. Biol. 1994; 242: 252-270Crossref PubMed Scopus (81) Google Scholar). To this solution of Gro-α was added microliter aliquots of a concentrated solution of H12 to achieve the following Gro-α (monomeric):H12 ratios: 20:1, 10:1, 4:1, 2:1, and 1:1. Addition of the initial H12 aliquot to the protein solution induced some opacity to the solution which, as with PF4-M2, became clear after heating to 50 °C for 30 min or allowing it to stand overnight at room temperature. When the Gro-α:H12 ratio reached 4:1 or greater, addition of more H12 induced no further cloudiness. Two-dimensional and three-dimensional NMR spectra were collected on a Varian Inova 600 spectrometer. H121H resonances were assigned by using a combination of TOCSY (21Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) (2048 × 256, 60-ms mixing time) and NOESY (22Jeener J. Meier B. Backman P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4550Crossref Scopus (4837) Google Scholar, 23Wider G. Macura S. Anil-Kumar Ernst R.R. Wüthrich K. J. Magn. Reson. 1984; 56: 207-234Google Scholar) (2048 × 256, 100-ms mixing time) experiments performed at 30 °C and at 50 °C. NMR results were similar to those reported previously by Pervin et al. (14Pervin A. Gallo C. Jandik K. Han X.-J. Linhardt R.J. Glycobiology. 1995; 5: 83-95Crossref PubMed Scopus (195) Google Scholar). In these two-dimensional NMR experiments, suppression of the water resonance was carried out using the WATERGATE sequence (24Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1012) Google Scholar). Two-dimensional NMR 15N gradient-enhanced HSQC (25Kay L.E. Keifer P. Saarinen S. J. Am. Chem. Soc. 1992; 114: 10663-10668Crossref Scopus (2429) Google Scholar) (2048 × 256) spectra were collected at various molar ratios of protein:H12 as noted above. The transmitter offset for the 1H dimension was set on the water resonance. The water signal was suppressed by applying selectively shaped pulses. Two-dimensional NMR data were processed off line using the programs Felix (Biosym/MSI, San Diego) and VNMR (Varian) by zero-filling to achieve 1024 × 1024 real data points and by using skewed-sinebell or Gaussian window functions. HCCH-TOCSY (26Clore G.M. Bax A. Driscoll P.C. Wingfield P.T. Gronenborn A.M. Biochemistry. 1990; 29: 8172-8184Crossref PubMed Scopus (192) Google Scholar) (64 × 48 × 1024, 15.6 ms mixing time) three-dimensional NMR experiments were acquired at 50 °C and were used to assign PF4-M2 side-chain resonances for the protein in the absence and in the presence of approximately 5-fold molar heparin excess. Previous 1H resonance assignments were used from data acquired on pure PF4-M2 (9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar). Assignments for the mixture were made under the assumption that 13C chemical shifts for side-chain carbons were nearly the same in both PF4-M2 free and H12-bound states. Gradient-enhanced TOCSY-HSQC (27Zhang O. Kay L.E. Olivier J.P. Forman-Kay J.D. J. Biomol. NMR. 1994; 4: 845-858Crossref PubMed Scopus (612) Google Scholar, 28Wijmenga S.S. van Mierlo C.P.M. Steensma E. J. Biomol. NMR. 1996; 8: 319-330Crossref PubMed Scopus (17) Google Scholar) (64 × 48 × 1024, 60-ms mixing time) three-dimensional NMR experiments were performed at 30 °C in order to complete 15N assignments of Gro-α.1H and 15N chemical shifts for Gro-α were essentially the same as those reported previously by Fairbrother et al. (20Fairbrother W.J. Reilly D. Colby T. Hesselgesser J. Horuk R. J. Mol. Biol. 1994; 242: 252-270Crossref PubMed Scopus (81) Google Scholar). The raw three-dimensional NMR data were zero-filled to 256 × 256 × 1024 and multiplied by a sinebell window function using VNMR software (Varian). Translational diffusion measurements were performed on a Varian Inova 500 NMR spectrometer by using the pulse field gradient (PFG) method as described previously (29Ilyina E. Roongta V. Pan H. Woodward C. Mayo K.H. Biochemistry. 1997; 36: 3383-3388Crossref PubMed Scopus (64) Google Scholar). Similar PFG-NMR studies have been done to assess the self-association and complexation of other protein systems (e.g. 30). The PFG longitudinal Eddy current delay pulse sequence (31Gibbs S.J. Johnson C.S. J. Magn. Reson. 1991; 93: 395-402Google Scholar) was used for all diffusion measurements. For unrestricted molecular diffusion in an isotropic medium, the amplitude of the NMR signal, A, normalized to the signal obtained in the absence of gradient pulses is related to the diffusion coefficient D by Equation 1.A(g2)=A(0)exp[−γ2g2δ2D(Δ−δ/3)]Equation 1 γ is the gyromagnetic ratio for the observed nucleus (1H in this case); g and δ are the magnitude and duration of the magnetic field gradient pulses, respectively, and Δ is the time between gradient pulses. Gradient strength was calibrated based on the known diffusion constant of water (2.3 × 10−5 cm2/s at 25 °C). Other parameters in Equation 1 were set as follows: δ = 4 ms, Δ = 34.2 ms. For each measurement, 11 NMR spectra were collected for different values of g and processed off-line by using a home-written VNMR macro. Diffusion constants for PF4-M2 and Gro-α were measured over a range of temperatures from 10 °C to 60 °C. Diffusion measurements of solutions of PF4 (30 mg/ml) and IL-8 (10 mg/ml) were used as references for tetramer and dimer sizes, respectively. The Stokes-Einstein equation,D=kBT/6πηR,Equation 2 where R is the macromolecular radius and η is the solution viscosity, was used to relate D to the apparent molecular weight Mapp. The apparent molecular weight for globular proteins is proportional to R 3, whereas for random coils, it is proportional to R 2 (32Cantor C.R. Schimmel P.R. Biophysical Chemistry. W. H. Freeman & Co., New York1980Google Scholar). From Equation 2, it follows that under the same conditions, diffusion coefficients for two different proteins related by their apparent molecular weights is shown by Equation 3.(D1/D2)=(M2app/M1app)aEquation 3 Here, the superscript a is 1/3 for compact proteins and 1/2 for random coils. According to this equation, a factor of 2 difference in molecular weights (e.g. monomer to dimer) results in a D 1/D 2 ratio of 1.26 to 1.41. This relationship, of course, is an approximation and works best for monomeric proteins having a spherical shape. The actual molecular shape and configuration of the aggregate are expected to affect the diffusion coefficient. Thus, using apparent molecular weights rather than experimentally measured diffusion coefficients can be a source of error. Therefore, these results are presented here in terms of diffusion coefficients. Circular dichroic (CD) spectra were measured on a Jasco JA-710 automatic recording spectropolarimeter coupled to a data processor. Curves were recorded digitally and fed through the data processor for signal averaging and base-line subtraction. Spectra were recorded at 30 °C in the presence of 10 mm potassium phosphate, over a 190–250-nm range using a 0.5-mm path length, thermally jacketed quartz cuvette. Temperature was controlled by using a NesLab water bath. Protein concentration was 0.03 mm. The scan speed was 20 nm/min. Spectra were signal-averaged eight times, and an equally signal-averaged solvent base line was subtracted. CD spectra were deconvoluted as described by Sreerama and Woody (33Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32Crossref PubMed Scopus (944) Google Scholar). Since α-chemokines PF4-M2 and Gro-α are known to bind heparin and heparin fragments relatively tightly (6Witt D.P. Lander A.D. Curr. Biol. 1994; 4: 394-400Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar), it was thought that mixing pure H12 and pure PF4-M2 or Gro-α would produce a complex of protein and GAG whose NMR spectral features would be similar to the sum of the individual species. The situation turned out to be not so simple, and the following sections report observations from diffusion, NMR, and CD measurements which catalog protein quaternary and tertiary structural changes induced by the presence of H12. To investigate how the size of PF4-M2 or Gro-α changed on binding H12, PFG-NMR self-diffusion measurements were performed at various protein (monomer):H12 ratios. For reference and calibration, Fig. 3 A plots the temperature dependence of diffusion coefficients, D, for native PF4, IL-8 dimer, Gro-α dimer, and PF4-M2 in the absence of H12. The temperature dependence of D for PF4, PF4-M2, and IL-8 is linear with apparent activation energies of 4.7, 5, and 5 kcal/mol, respectively, and follows the activation energy expected for the self-diffusion of water, 4.8 kcal/mol. Thus, one can be confident that the aggregate state of these proteins remains essentially unchanged over this temperature range and that the distribution is homogeneous, i.e. multiple oligomerization states either are not present or are minimal. Therefore, it is reasonable to conclude that, as indicated in the literature, aggregation states for native PF4 (34Mayo K.H. Chen M.J. Biochemistry. 1989; 28: 9469-9478Crossref PubMed Scopus (85) Google Scholar) and IL-8 (35Clore G.M. Appella E. Yamada M. Matsushima K. Gronenborn A.M. J. Biol. Chem. 1989; 264: 18907-18911Abstract Full Text PDF PubMed Google Scholar) are tetramer and dimer, respectively, at these concentrations. The temperature dependence of D for pure Gro-α at 10 mg/ml, although linear with an apparent activation energy value of 4.3 kcal/mol, does deviate more than expected for a single size species. Since Gro-α and IL-8 have nearly the same monomer molecular weight and are known to form the same type of dimers (20Fairbrother W.J. Reilly D. Colby T. Hesselgesser J. Horuk R. J. Mol. Biol. 1994; 242: 252-270Crossref PubMed Scopus (81) Google Scholar,35Clore G.M. Appella E. Yamada M. Matsushima K. Gronenborn A.M. J. Biol. Chem. 1989; 264: 18907-18911Abstract Full Text PDF PubMed Google Scholar), their dimers should display nearly the same diffusion coefficients. This is not the case with D values for Gro-α being larger than those for dimeric IL-8 and the difference in their apparent activation energies being 0.7 kcal/mol. Moreover, the temperature dependence for changes in aggregate state distributions for homologous α-chemokines PF4 (34Mayo K.H. Chen M.J. Biochemistry. 1989; 28: 9469-9478Crossref PubMed Scopus (85) Google Scholar) and NAP-2 (neutrophil activating peptide-2) (16Yang Y. Mayo K.H. Daly T.J. Barry J.K. La Rosa G.L. J. Biol. Chem. 1994; 269: 20110-20118Abstract Full Text PDF PubMed Google Scholar) is small, suggesting a primarily entropy-driven process of self-association. This is consistent with the observed minimal change in activation energies for partially dissociated Gro-α. These diffusion data, therefore, indicate that Gro-α at this concentration is partly dissociated, more so at lower temperature as also observed with other α-chemokines. At 30 °C, a D value of about 15 × 10−7 cm2/s is expected for compactly folded, monomeric Gro-α, assuming the ratio of monomer/dimer diffusion coefficients is similar to that of tetramer/dimer. This would indicate that, at 30 °C, this preparation of Gro-α is about 44% dimeric. For PF4-M2, diffusion coefficients over the temperature range 10–40 °C are the same as those for IL-8, indicating that PF4-M2 is primarily dimeric under these solution conditions (6 mg/ml). This was unexpected since earlier studies (9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar) indicated that PF4-M2 at 6 mg/ml would be a mix of mostly dimers and tetramers. Moreover,13C and 15N NMR relaxation data (T1, T2, and NOE) 2V. Roongta, E. Ilyina, V. Daragan, and K. H. Mayo, unpublished results. yield rotational correlation times also consistent with PF4-M2 being primarily dimeric. These data contradict our earlier results regarding the aggregation states of PF4-M2 (9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar). Originally, the equilibrium between monomer, dimer, and tetramer states of native PF4 (34Mayo K.H. Chen M.J. Biochemistry. 1989; 28: 9469-9478Crossref PubMed Scopus (85) Google Scholar) and PF4-M2 (9Mayo K.H. Roongta V. Ilyina E. Millius R. Baker S. Quinlan C. La Rosa G. Daly T.J. Biochemistry. 1995; 34: 11399-11409Crossref PubMed Scopus (53) Google Scholar) was interpreted via analysis of integrated intensities for three different Tyr-60 ring proton resonances, which arise from slow monomer-dimer-tetramer exchange on the chemical shift time scale. In the present 13C/15N-enriched PF4-M2 preparation, only the Tyr-60 ring proton resonances corresponding to dimer and monom" @default.
• W2038952850 created "2016-06-24" @default.
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• W2038952850 title "Heparin Dodecasaccharide Binding to Platelet Factor-4 and Growth-related Protein-α" @default.
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