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• W1998773453 abstract "15N NMR relaxation data have been used to characterize the backbone dynamics of the human acidic fibroblast growth factor (hFGF-1) in its free and sucrose octasulfate (SOS)-bound states. 15N longitudinal (R1), transverse (R2) relaxation rates and {1H}-15N steady-state nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25 °C) for all resolved backbone amide groups using 1H- detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time (τm) for the free and SOS-bound forms were estimated to be 10.4 ± 1.07 and 11.1 ± 1.35 ns, respectively. Titration experiments with SOS reveals that the ligand binds specifically to the C-terminal domain of the protein in a 1:1 ratio. Binding of SOS to hFGF-1 is found to induce a subtle conformational change in the protein. Significant conformational exchange (Rex) is observed for several residues in the free form of the protein. However, in the SOS-bound form only three residues exhibit significant Rex values, suggesting that the dynamics on the micro- to millisecond time scale in the free form is coupled to the cis-trans-proline isomerization. hFGF-1 is a rigid molecule with an average generalized parameter (S2) value of 0.89 ± 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S2 = 0.94 ± 0.02) of the hFGF-1 molecule. However, the segment comprising residues 103–111 shows increased flexibility in the presence of SOS. Significant correlation is found between residues that show high flexibility and the putative receptor binding sites on the protein. 15N NMR relaxation data have been used to characterize the backbone dynamics of the human acidic fibroblast growth factor (hFGF-1) in its free and sucrose octasulfate (SOS)-bound states. 15N longitudinal (R1), transverse (R2) relaxation rates and {1H}-15N steady-state nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25 °C) for all resolved backbone amide groups using 1H- detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time (τm) for the free and SOS-bound forms were estimated to be 10.4 ± 1.07 and 11.1 ± 1.35 ns, respectively. Titration experiments with SOS reveals that the ligand binds specifically to the C-terminal domain of the protein in a 1:1 ratio. Binding of SOS to hFGF-1 is found to induce a subtle conformational change in the protein. Significant conformational exchange (Rex) is observed for several residues in the free form of the protein. However, in the SOS-bound form only three residues exhibit significant Rex values, suggesting that the dynamics on the micro- to millisecond time scale in the free form is coupled to the cis-trans-proline isomerization. hFGF-1 is a rigid molecule with an average generalized parameter (S2) value of 0.89 ± 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S2 = 0.94 ± 0.02) of the hFGF-1 molecule. However, the segment comprising residues 103–111 shows increased flexibility in the presence of SOS. Significant correlation is found between residues that show high flexibility and the putative receptor binding sites on the protein. human acidic fibroblast growth factor sucrose octasulfate fibroblast growth factor FGF receptor heparan sulfate proteoglycans nuclear Overhauser effect Human acidic fibroblast growth factor (hFGF-1)1 is a 16-kDa protein, with a wide array of biological activities such as morphogenesis development, angiogenesis, and wound healing (1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 2Bascilico C. Mostacelli D. Adv. Cancer Res. 1992; 59: 115-175Crossref PubMed Scopus (1049) Google Scholar, 3Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (589) Google Scholar, 4Pineda-Lucena A. Jimenez M.A. Lozano R.M. Nieto J.S. Santoro J. Rico M. Gimenez-Gallego G. J. Mol. Biol. 1994; 242: 81-89Crossref PubMed Scopus (45) Google Scholar). The secondary structural elements in the protein include 12 β-strands arranged antiparallel into a β-barrel structure (5Pineda-Lucena A. Jimenez M.A. Lozano R.M. Nieto J.S. Santoro J. Rico M. Gimenez-Gallego G. J. Mol. Biol. 1996; 264: 162-178Crossref PubMed Scopus (51) Google Scholar, 6Zhu X. Komiya H. Chirino A. Faham S. Fox G.M. Arakawa T. Hsu B. Rees D.C. Science. 1991; 251: 90-93Crossref PubMed Scopus (327) Google Scholar, 7Ogura K. Nagata K. Hatanaka H. Habuchi H. Kimata K. Tati S. Raveru M.W. Jaye M. Schlessinger J. Inagaki F. J. Biomol. NMR. 1999; 13: 11-24Crossref PubMed Scopus (51) Google Scholar). hFGF-1 requires two kinds of receptors as follows: low affinity receptors that are heparan sulfate proteoglycans (HSPGs) and the high affinity receptors (FGFRs) that are transmembrane tyrosine kinases (2Bascilico C. Mostacelli D. Adv. Cancer Res. 1992; 59: 115-175Crossref PubMed Scopus (1049) Google Scholar, 4Pineda-Lucena A. Jimenez M.A. Lozano R.M. Nieto J.S. Santoro J. Rico M. Gimenez-Gallego G. J. Mol. Biol. 1994; 242: 81-89Crossref PubMed Scopus (45) Google Scholar, 8Herr A.B. Ornitz D.M. Sasisekharan R. Venkataraman G. Waksman G. J. Biochem. ( Tokyo ). 1997; 272: 16382-16389Google Scholar, 9Faham S. Linhardt R.J. Rees D.C. Curr. Opin. Struct. Biol. 1998; 8: 578-586Crossref PubMed Scopus (138) Google Scholar, 10Springer B.A. Pantoliano M.W. Barbera F.A. Gunyuzhu P.L. Thompson L.D. Herbin W.F. Rosenfield S.A. Book G.W. J. Biol. Chem. 1994; 269: 26879-26884Abstract Full Text PDF PubMed Google Scholar). The FGFRs are composed of an extracellular ligand binding portion consisting of three immunoglobulin-like domains (D1, D2, and D3), a transmembrane helix, and a cytoplasmic domain that contains the tyrosine kinase activity (11Plotnikov A. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). The HSPGs are contemplated to bind and dimerize FGF and present the growth factor molecules to the receptor to form a ternary FGF·HSPG·FGFR complex (9Faham S. Linhardt R.J. Rees D.C. Curr. Opin. Struct. Biol. 1998; 8: 578-586Crossref PubMed Scopus (138) Google Scholar, 11Plotnikov A. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). The exact role of heparin/heparan sulfate in the activation of the hFGF-1 signaling pathway is still not clear. Some studies have shown that heparin binds to multiple FGF molecules and induces their oligomerization (8Herr A.B. Ornitz D.M. Sasisekharan R. Venkataraman G. Waksman G. J. Biochem. ( Tokyo ). 1997; 272: 16382-16389Google Scholar). Oligomerization of hFGF has been proposed as a prerequisite for binding and activation of the receptor (8Herr A.B. Ornitz D.M. Sasisekharan R. Venkataraman G. Waksman G. J. Biochem. ( Tokyo ). 1997; 272: 16382-16389Google Scholar). In contrast, results of other studies suggest a protective role for heparin/heparan sulfate (12Mach H. Middaugh C.R. Arch. Biochem. Biophys. 1994; 309: 36-42Crossref PubMed Scopus (9) Google Scholar, 13Burke C.J. Volkin D.B. Mach H. Middaugh C.R. Biochemistry. 1993; 32: 6419-6426Crossref PubMed Scopus (58) Google Scholar, 14Dabora J.M. Sanyal G. Middaugh C.R. J. Biol. Chem. 1991; 266: 23637-23640Abstract Full Text PDF PubMed Google Scholar). Binding of hFGF to the glycosaminoglycans is shown to protect FGFs against proteolytic digestion and heat- and acid-induced unfolding (15Sanz J.M. Gallego G.G. Eur. J. Biochem. 1997; 246: 328-335Crossref PubMed Scopus (28) Google Scholar, 16Blaber S.I. Culajay J.F. Khurana A. Blaber M. Biophys. J. 1999; 77: 470-477Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Gaspodarowicz D. Cheng J. J. Cell. Physiol. 1986; 128: 475-484Crossref PubMed Scopus (677) Google Scholar). The high affinity tyrosine kinase activation and signaling are known to be dependent of FGF-induced dimerization. Dimerization of the receptor molecules is believed to lead to juxtaposition of the cytoplasmic domains and subsequent transphosphorylation on tyrosine residues in the cytoplasmic domain (11Plotnikov A. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Crystal structure of the FGF·FGFR complexed to its ligand (FGF) shows that the FGF·FGFR complex is partially stabilized by direct FGFR-FGFR interactions and by interactions between FGF and FGFR (11Plotnikov A. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 18Plotnikov A. Hubbard S.R. Schlessinger J. Mohammadi M. Cell. 2000; 101: 413-424Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Interestingly, no direct FGF-FGF interactions were observed in the FGF·FGFR complex. The FGF·FGFR complex structure has a positively charged canyon and is believed to represent the heparin-binding site (11Plotnikov A. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). However, to date the three-dimensional structure of the FGF·HSPG·FGFR ternary complex has not been solved. Protein mobility plays an important role in the recognition process involved in protein-receptor, antigen-antibody, and enzyme-substrate interaction. One useful method to study motion of protein molecules involves the analysis of NMR relaxation processes (19Palmer III, A.G. Rance M. Wright P.E. J. Am. Chem. Soc. 1991; 113: 4371-4380Crossref Scopus (595) Google Scholar, 20Ye J. Mayer K.L. Stone M.J. J. Biomol. NMR. 1999; 15: 115-124Crossref PubMed Scopus (32) Google Scholar). With the advent of inverse detection methods, it has been feasible to analyze the backbone dynamics of proteins at a residue level using 15N relaxation measurements (21Pascal S.M. Yamazaki T. Singer A.U. Kay L.E. Kay J.D. Biochemistry. 1995; 34: 11353-11362Crossref PubMed Scopus (58) Google Scholar, 22Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (883) Google Scholar, 23Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1774) Google Scholar, 24Stone M.J. Chandrasekhar K. Holmgren A. Wright P.E. Dyson H.J. Biochemistry. 1993; 32: 426-435Crossref PubMed Scopus (137) Google Scholar, 25Stivers J.T. Abeygunawardana C. Mildvan A.S. Biochemistry. 1996; 35: 16036-16047Crossref PubMed Scopus (104) Google Scholar, 26Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay I.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (1992) Google Scholar). In this background, we analyze the backbone dynamics of free hFGF-1 and its complex with a heparin structural analog sucrose octasulfate (SOS) using 1H-15N NMR techniques. Heparin-Sepharose was obtained from Amersham Pharmacia Biotech. Labeled 15NH4Cl was purchased from Cambridge Isotope Laboratories. SOS was purchased from Toronto Research Chemicals. All other chemicals used were of high quality analytical grade. Unless mentioned, all solutions were made in 100 mmphosphate buffer (pH 7.0) containing 100 mm of ammonium sulfate. All experiments were performed at 25 °C. Residues are numbered as per their position in the primary structure of the 154 amino acid hFGF-1. Expression vector for the truncated form of the human FGF-1 (hFGF-1, residues 15–154) was constructed and inserted between theNdeI and BamHI restriction sites in pET20b. The plasmid containing the hFGf-1 insert was transformed intoEscherichia coli BL21(DE3)pLysS. The expressed protein was purified on heparin-Sepharose using a NaCl gradient (0–1.5m). The protein was desalted by ultrafiltration using an Amicon set up. The homogeneity of the protein was assessed using SDS-polyacrylamide gel electrophoresis. The authenticity of the sample was further verified by electron spray-mass analysis. The concentration of the protein was estimated from the extinction coefficient value of the protein at 280 nm. Uniform15N isotope labeling was achieved using M9 minimal medium containing 15NH4Cl. In order to realize maximal expression yields, the composition of the M9 medium was modified by the addition of a mixture of vitamins. The expression host strain E. coli BL21(DE3)pLysS is a vitamin B1-deficient host, and hence the medium was supplemented with thiamine (vitamin B1). Protein expression yields were in the range of 25–30 mg/liter of the isotope-enriched medium. The extent of 15N labeling was verified by electron spray-mass analysis. 15N longitudinal transverse relaxation and NOE data were collected on Varian Inova 500 MHz and Bruker DMX 600 MHz NMR spectrometers. Protein samples for relaxation measurements were dissolved at a concentration of ∼1.5 mm in 100 mm phosphate buffer (pH 6.5) (containing 100 mm ammonium sulfate) prepared using 10% D2O and 90% H2O (at 25 °C). A 1:1 hFGF-1 and SOS complex was obtained by titration of the protein with SOS. 15N chemical shifts were referenced using the consensus ratio of 0.0101329118. All spectra were processed on a Silicon Graphics workstation using the UXNMR and AURELIA softwares. 15N longitudinal (R1) and transverse (R2) relaxation rates and heteronuclear {1H}-15N NOE were measured using two-dimensional 1H-15N correlation pulse sequences (26Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay I.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (1992) Google Scholar). For the R1 and R2 experiments (at 500 and 600 MHz), 16 and 24 scans, respectively, were collected pert1 point, and for the {1H}-15N NOE experiments, 32 scans pert1 point were acquired. The 15N R1 and R2 values at 600 MHz were obtained using 8 delays, namely 40, 60, 140, 240, 360, 520, 720, and 1200 ms for the R1 experiment and 16.2, 32.4, 48.6, 64.8, 81, 97.2, 113.4, and 129.6 ms for the R2 experiments. The 15N R1 and R2 values at 500 MHz were obtained using 8 delays (t) at 40, 60, 140, 240, 360, 720, and 1200 ms for the R1 experiment and 10, 30, 50, 70, 110, 130, and 150 ms for the R2 experiments. The {1H}-15N steady-state NOE values at 500 and 600 MHz were obtained by recording spectra with and without a proton presaturation period (6 s) applied before the start of the1H-15N HSQC experiment. The NOE spectra were acquired in an interleaved manner, in which each individual FID was collected with and without proton presaturation. All spectra were processed identically on a Silicon Graphics workstation using the AURELIA and UXNMR softwares. The peak heights in the two-dimensional spectra were measured using the peak picking subroutine. The R1 and R2 values were determined by fitting the peak heights as a function of the relaxation interval (t) to a two-parameter (Io and R1,2) exponential decay function, I(T) = Io exp (−R1,2 t), using a nonlinear least squares analysis (27Press W.H. Flannery B.P. Teukolsky S.A. veHerling W.T. Numerical Recipes in Fortran. 2nd Ed. Cambridge University Press, UK1992: 48-67Google Scholar, 28Leatherbarrow R.J. Graft , Version 3.0. Erithacus Softwares Ltd, Staines, UK1992Google Scholar). The average uncertainty values of R1,2 were determined from three data sets. The steady-state NOE values at 500 and 600 MHz were determined from the ratios of the peak intensities with and without presaturation. The standard deviation of the NOE was determined from the root mean square value of the background noise in the spectra. In all, three separate NOE data sets were collected at both 500 and 600 MHz. Analysis of R1, R2, and NOE data followed the procedure outlined by Mandel et al. (29Mandel A.M. Akke M. Palmer III, A.G. J. Mol. Biol. 1995; 246: 144-163Crossref PubMed Scopus (896) Google Scholar). The relaxation rates were analyzed using the model free formalism proposed by Lipari and Szabo (30Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4546-4559Crossref Scopus (3342) Google Scholar, 31Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4559-4570Crossref Scopus (1850) Google Scholar) and extended by Clore et al. (32Clore G.M. Driscoll P.C. Wingfield P.T. Gronenborn A.M. Biochemistry. 1990; 29: 7387-7401Crossref PubMed Scopus (516) Google Scholar). The spectral density function, J(ω) is modeled as shown in Equations 1and 2,J(ω)=2/5[{S2τm/1+(ωτm) 2}+{(1−Sf2)τf′/1Equation 1 +(ωτf′) 2}+{(Sf2−S2)τs′/1+(ωτs′) 2}]whereτf′=τfτm/(τf+τm),τs′=τsτm/(τs+τm)Equation 2 τm is the overall rotational correlation time of the molecule. τf is the effective correlation time for internal motion on a fast time scale (τf < 100 ps); τs is the effective correlation time for internal motions on a slow time scale (τe<τs<τm); S2 = S2f. S2s, is the generalized order parameter characterizing the amplitude of the internal motions, and S2f and S2s are the order parameters for the internal motions on the fast and slow time scales, respectively. The order parameters specify the degree of spatial restriction of the 1H-15N bond vector, with values ranging from zero for isotropic internal motions to unity for completely restricted motion and represent dynamics on the pico- to nanosecond time scale. Five alternative models for the spectral density functionJ(ω) were used to fit the experimental values of R1 and R2 and steady-state {1H}-15N NOEs (32Clore G.M. Driscoll P.C. Wingfield P.T. Gronenborn A.M. Biochemistry. 1990; 29: 7387-7401Crossref PubMed Scopus (516) Google Scholar). All the models are based on the spectral density function of Lipari and Szabo (31Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4559-4570Crossref Scopus (1850) Google Scholar, 32Clore G.M. Driscoll P.C. Wingfield P.T. Gronenborn A.M. Biochemistry. 1990; 29: 7387-7401Crossref PubMed Scopus (516) Google Scholar), which in its simplest form includes a generalized order parameter, S2, and an effective correlation time, τe. The first model includes only the generalized order parameter (S2) with τe = 0, and the second model includes both S2 and τe. The effects of millisecond-microsecond time scale motions (Rex) are included in the third and fourth models. In model 3, S2 and Rex are fit, and S2, Rex and τewere fit in model 4. The fifth model includes two high frequency motions separated by at least 1 order of magnitude. The various models and the number of residues fitting using different models are presented in Table I.Table ISpectral density functions used in the analysis of the 15 N relaxation dataModelSpectral density functionOptimized parametersNo. of residues for free hFGF-1No. of residues for SOS-bound hFGF-11J(ω) = 2/5[S2τm/(1 + (ωτm)2)]S222142J(ω) = 2/5[S2τm/(1 + (ωτm)2) + (1 − S2)τ′e/(1 + (ωτ′e)2)]S23858τe3J(ω) = 2/5[S2τm/(1 + (ωτm)2)]1/],T2obs = 1/T2 + RexS280Rex4J(ω) = 2/5[S2τm/(1 + (ωτm)2) + (1 − S2)τ′e/(1 + (ωτ′e)2)], 1/T2obs = 1/T2 + RexS2123τeRex5J(ω) = 2/5[S2τm/(1 + (ωτm)2) + S2f(1 − S2s)τ′s/(1 + (ωτ′s)2)]Sf2, Ss21324τeτ′e = τmτe/(τm + τe) τ′s = τmτs/(τm + τs), S2 = S2fS2s Open table in a new tab SOS is a disaccharide structural analogue of heparin and has been shown to possess anti-ulcer properties (33Volkin D.B. Verticelli A.M. Marfia K.E. Burke C.J. Mach H. Middaugh C.R. Biochim. Biophys. Acta. 1993; 1203: 18-26Crossref PubMed Scopus (50) Google Scholar). SOS has been shown to protect hFGF-1 from pH-induced and thermal-induced inactivation. SOS has been shown to bind to heparin-binding sites on hFGF-1 and stimulate the angiogenic and mitogenic activities of the protein (33Volkin D.B. Verticelli A.M. Marfia K.E. Burke C.J. Mach H. Middaugh C.R. Biochim. Biophys. Acta. 1993; 1203: 18-26Crossref PubMed Scopus (50) Google Scholar). In vitro studies have revealed that unlike heparin, which oligomerizes the protein, SOS maintains the hFGF-1 molecule in a monomeric state (3Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (589) Google Scholar). Thus, SOS effectively mimics the structural and functional properties of heparin and provides a scope to understand the effects of heparin on the backbone dynamics of hFGF-1 using 15N NMR relaxation methods. The binding of SOS to hFGF-1 was followed by collecting a series of1H-15N HSQC spectra at varying protein to ligand ratios (1:0 to 1:1.5 ratio). All the cross-peaks in the spectra were assigned based on the previously published 1H and15N NMR assignments of hFGF-1 (7Ogura K. Nagata K. Hatanaka H. Habuchi H. Kimata K. Tati S. Raveru M.W. Jaye M. Schlessinger J. Inagaki F. J. Biomol. NMR. 1999; 13: 11-24Crossref PubMed Scopus (51) Google Scholar). The1H-15N HSQC spectrum complexed to SOS at a 1:1 ratio shows that the chemical shift of most of the cross-peaks are not altered appreciably upon addition of SOS (Fig. 1). The cross-peaks that show maximal chemical shift perturbation are located at the C-terminal end spanning residues 126–137 (Fig. 2). It should be mentioned that increase of protein to SOS ratio beyond 1:1 causes no further significant perturbation in the chemical shifts of cross-peaks in the 1H-15N HSQC spectrum of the protein, implying that the binding of SOS to protein is specific and the binding sites are restricted to definite regions(s) of the structure of hFGF-1. This observation is quite consistent with the crystal structure of the hFGF-1·SOS complex, whereas the ligand (SOS) has been shown to bind to a cluster of positively charged residues located in the C-terminal domain (residues 120–142) of the protein (6Zhu X. Komiya H. Chirino A. Faham S. Fox G.M. Arakawa T. Hsu B. Rees D.C. Science. 1991; 251: 90-93Crossref PubMed Scopus (327) Google Scholar). It appears that binding of SOS to the residues at the C-terminal domain decreases the charge repulsion encountered by the cationic residues located at close proximity in this region (in the C-terminal domain) and consequently increases the thermodynamic stability of the protein.Figure 2Weighted average (of 15N and1H) chemical shift perturbation (Δδ = √(δH2 + 0.2 (δ15N)2) of residues in hFGF-1 upon complex formation with SOS (at a protein to SOS ratio of 1:1). It could be discerned that the residues 126–143 at the C-terminal end (indicated by the bracket) display maximal chemical shift perturbation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The relaxation rate parameters R1, R2, and NOE were obtained by analysis of proton-detected 15N and 1H heteronuclear correlation spectra of the free and SOS-bound form of hFGF-1. Reliable quantitation of peak intensities were possible for 110 out of the expected 140 15NH backbone resonances in both the free and SOS-bound forms of hFGF-1. The residues for which the relaxation data could not be acquired include 7 prolines, or those whose resonances are highly overlapped (7 residues) or too weak to quantify accurately (16 residues). 15N relaxation data were collected at 25 °C at 500 and 600 MHz field strengths. In all cases, the decay in intensity for all residues is found to be strictly exponential for both the 15N R1 and R2 data (at both 500 MHz and 600 MHz). R1 and R2 values are sensitive to different motional frequencies. R1 values provide information about motional properties with a frequency of approximately 108–1012 s−1, whereas R2 values, in addition to depending on motions occurring at these frequencies, are also sensitive to dynamics on the microsecond-millisecond time scale. Hence, by measuring both R1 and R2, it is feasible to obtain dynamic information over a large motional regime. The R1 and R2 values of hFGF-1 in its free and SOS-bound forms as a function of the residue number are shown in Fig. 3, panels A and B. The R1 values for the free form of hFGF-1 are remarkably uniform over most of the molecule with mean values of 1.17 ± 0.07 and 1.61 ± 0.08 s−1 at 500 and 600 MHz, respectively. However, significantly greater than average R1 values are seen for several residues in the free form of hFGF-1. 18 residues show an R1 value greater than 1.25 s−1. Three residues, namely Gly-76 (1.38 ± 0.13 s−1), Glu-118 (1.33 ± 0.06 s−1), and Ser-132 (1.43 ± 0.05 s−1), exhibit extraordinarily large R1 values (Fig. 3, panel A). Although the residues exhibiting noticeably higher R1 values (>1.2 s−1) are scattered throughout the hFGF-1 molecule, most of the residues located in the stretch spanning Tyr-78 to Leu-86 exhibit large R1 values indicating a restricted mobility in this region of the protein molecule in its free state. It is interesting to note that 10 residues display R1 values lower than the average R1 values (R1 <1.10 s−1). Some of these residues that show increased mobility are known to constitute the binding sites for heparin in the protein (Fig. 3, panel A). The average R1 (at 600 MHz) of the protein molecule almost remains unchanged (average R1 = 1.17 ± 0.07 s−1) upon complex formation with SOS, implying an overall similarity in the mobility of residues in the protein in the free and ligand-bound forms. Nine residues in the SOS complexed form reveal R1 values significantly higher (>1.25 s−1) than the trimmed average (Fig. 3,panel B). Most of the residues comprising the heparin-binding site show marginally increased R1 values indicating a decrease in the nanosecond time scale motion (of these residues) upon binding to SOS. Although R1 and R2 are influenced by motions over a range of time scales, the transverse relaxation rate, R2, is more sensitive to lower frequency (nanosecond) motions and also reflects contributions from slower millisecond or microsecond exchange processes that may cause line broadening in the NMR spectrum. The R2 values for the free form of hFGF-1 are uniform for most of the molecules, with mean values of 13.07 ± 2.1 and 15.06 ± 2.9 s−1 at 500 and 600 MHz, respectively. The R2 values at 600 MHz are consistently higher than the values at 500 MHz. There are several residues in the free form of the protein that display R2relaxation values greater than the trimmed average (Fig. 3, panel A). Most notable among them are Gly-34 (21.19 ± 1.65 s−1), Glu-54 (25.72 ± 0.81 s−1), His-55 (20.27 ± 1.1 s−1), His-107 (32.07 ± 0.38 s−1), Lys-115 (18.41 ± 0.51 s−1), Glu-118 (17.76 ± 0.37 s−1), Trp-121 (20.02 ± 1.28 s−1), and Leu-147 (19.14 ± 1.90 s−1). Similarly, many residues at the N- and C-terminal ends in the free state of the protein show R2values significantly lower than the trimmed average (Fig. 3,panel A). In addition, the residues in the segments spanning Ala-62 to Gly-66, Leu-87 to Asn-94, and Tyr-108 to Ser-113 (with the exception of Asn-109) show R2 values lower also than the trimmed average value (Fig. 3, panel A). The average R2 relaxation value of hFGF-1 is found to increase upon binding to SOS (average R2 = 17.37 ± 3.16 s−1 at 600 MHz, Fig. 3, panel B). The general trends of the R2 values in the free and ligand complexed states of the protein are similar. However, in all cases, the R2 values of residues in the presence of SOS are higher than that observed in the ligand free state of the protein. In the SOS-complexed form, many residues in the C-terminal segment (Arg-136, Ala-143, Leu-135, and Leu-147) show R2 values significantly greater than >19 s−1, the trimmed average (Fig. 3, panel B). Interestingly, these residues in the free state of the protein possess R2 values that are in the range of the average R2 relaxation rate (in the free form of the protein). In general, residues exhibiting low R2 values are believed to be involved in internal motions on the nano- and picosecond time scale. In this background, the residues at the N- and C-terminal ends that have low R2 rates appear to undergo rapid, nanoseconds-picoseconds internal dynamics. It is interesting to note that there are residues distributed in three segments, namely Ala-62 to Gly-66, Leu-84 to Asn-94, and Tyr-108 to Ser-113 (with the exception of Asn-109) which are in rapid motions on the nano- or picosecond time scale. Some of the residues involved in these segments of the protein have been shown to represent the contact sites for the hFGF-1/receptor recognition (11Plotnikov A. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 18Plotnikov A. Hubbard S.R. Schlessinger J. Mohammadi M. Cell. 2000; 101: 413-424Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). However, a similar analysis of the residues exhibiting exceptionally large R2 could not be provided because anomalously large R2 rates could be due to either large contributions from conformational exchange dynamics (Rex) on the micro- to millisecond time scales or due to anisotropic rotational diffusion of the protein (to be discussed later). {1H}-15N NOE data such as the relaxation of longitudinal and transverse magnetization of backbone15N nuclei are sensitive to motions on a nanosecond to picosecond time scale. The {1H}-15N NOE is typically most sensitive to higher frequency motions of the backbone, with values near 1.0 indicating a lack of such motions, and lower values indicating increased local flexibility of the polypeptide (24Stone M.J. Chandrasekhar K. Holmgren A. Wright P.E. Dyson H.J. Biochemistry. 1993; 32: 426-435Crossref PubMed Scopus (137) Google Scholar). The average {1H}-15N steady-state NOE values estimated for free hFGF-1 at 600 and 500 MHz are 0.79 ± 0.18 and 0.73 ± 0.21, respectively. Most of the residues, which show low R2 values (lower than the trimmed ave" @default.
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