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. 2009 Oct 26;284(52):36535–36546. doi: 10.1074/jbc.M109.037267

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© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — NMR structural analyses of sA27-aa and K3A3. A, two-dimensional ¹H-¹⁵N HSQC NMR spectra of the parent sA27-aa. B, HSQC spectrum of the scrambled HBS sequence mutant K3A3 at pH 5.0. The respective GAG-binding sequences are shown above the spectrum with the mutated residues underlined. Due to protein self-assembly, only the residues of the flexible HBS were observable (for details, see the text). The respective sequential assignments were achieved on the basis of three-dimensional HNCA and HN(CO)CA NMR experiments (43), and the inter- and intra-residue chemical shift correlations were determined (supplemental Table S2). Residues are boxed if a substantial chemical shift difference was found in the spectra. Single letter abbreviations for amino acids are used. The numbering is based on the sequence of wild type A27. C, ¹H^N; D, ¹³C^α; and E, ¹³CO chemical shift propensity index analysis of the GAG-binding sequences of sA27-aa and K3A3. The chemical shift index was determined as (δ-δ_random)/(δ_β-δ_random), where δ is the experimental chemical shift and δ_β and δ_random the chemical shift values reported in the literature for a β-structure and random coil, respectively. In the KKPE-binding motif of sA27-aa, the propensity index is around +0.5, suggesting a β-turn conformation, whereas in K3A3, no such structure is seen. The sequence numbering for sA27-aa (single letter code) is shown on the horizontal axis. F, two-dimensional ¹H-¹⁵N HSQC NMR spectra of sA27-aa interacting with HP, respectively. Spectrum of 0.6 mm [¹⁵N]sA27-aa protein without HP (red) is overlaid with the spectrum of the protein after the addition of HP (blue). In sA27-aa, for residues such as Ala²⁵, Lys²⁶, Lys²⁷, Glu²⁹, and Ala³⁰, chemical shift perturbations are greater than 0.01 ppm, suggesting that these residues are involved in specific HP binding. For other residues, namely Ser²¹, Glu³³, Ala³⁴, chemical shifts were unperturbed, suggesting that these are not involved in the HP binding. Insets showed expanded regions for some of these residues. G, two-dimensional ¹H-¹⁵N HSQC NMR spectra of K3A3 interacting with HP, respectively. Spectrum of 0.6 mm [¹⁵N]K3A3 protein without HP (red) is overlaid with the spectrum of the protein after the addition of HP (blue). Weight ratios of protein to HP are 1:0 (red), and 1:5 (blue).

FIGURE 3. — NMR structural analyses of sA27-aa and K3A3. A, two-dimensional ¹H-¹⁵N HSQC NMR spectra of the parent sA27-aa. B, HSQC spectrum of the scrambled HBS sequence mutant K3A3 at pH 5.0. The respective GAG-binding sequences are shown above the spectrum with the mutated residues underlined. Due to protein self-assembly, only the residues of the flexible HBS were observable (for details, see the text). The respective sequential assignments were achieved on the basis of three-dimensional HNCA and HN(CO)CA NMR experiments (43), and the inter- and intra-residue chemical shift correlations were determined (supplemental Table S2). Residues are boxed if a substantial chemical shift difference was found in the spectra. Single letter abbreviations for amino acids are used. The numbering is based on the sequence of wild type A27. C, ¹H^N; D, ¹³C^α; and E, ¹³CO chemical shift propensity index analysis of the GAG-binding sequences of sA27-aa and K3A3. The chemical shift index was determined as (δ-δ_random)/(δ_β-δ_random), where δ is the experimental chemical shift and δ_β and δ_random the chemical shift values reported in the literature for a β-structure and random coil, respectively. In the KKPE-binding motif of sA27-aa, the propensity index is around +0.5, suggesting a β-turn conformation, whereas in K3A3, no such structure is seen. The sequence numbering for sA27-aa (single letter code) is shown on the horizontal axis. F, two-dimensional ¹H-¹⁵N HSQC NMR spectra of sA27-aa interacting with HP, respectively. Spectrum of 0.6 mm [¹⁵N]sA27-aa protein without HP (red) is overlaid with the spectrum of the protein after the addition of HP (blue). In sA27-aa, for residues such as Ala²⁵, Lys²⁶, Lys²⁷, Glu²⁹, and Ala³⁰, chemical shift perturbations are greater than 0.01 ppm, suggesting that these residues are involved in specific HP binding. For other residues, namely Ser²¹, Glu³³, Ala³⁴, chemical shifts were unperturbed, suggesting that these are not involved in the HP binding. Insets showed expanded regions for some of these residues. G, two-dimensional ¹H-¹⁵N HSQC NMR spectra of K3A3 interacting with HP, respectively. Spectrum of 0.6 mm [¹⁵N]K3A3 protein without HP (red) is overlaid with the spectrum of the protein after the addition of HP (blue). Weight ratios of protein to HP are 1:0 (red), and 1:5 (blue).