ABCMdb

ABCC8 p.Phe48Lys

Predicted by SNAP2:	A: N (53%), C: N (66%), D: D (71%), E: D (53%), G: D (59%), H: D (53%), I: N (78%), K: D (66%), L: N (82%), M: N (72%), N: D (63%), P: D (71%), Q: D (53%), R: D (66%), S: N (53%), T: N (61%), V: N (57%), W: N (53%), Y: N (57%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: N,

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Publications
[hide] Investigation of the role of a surface patch in th... Biochim Biophys Acta. 1999 Aug 17;1433(1-2):159-69. Couture MM, Auger M, Rosell F, Mauk AG, Boubour E, Lennox RB, Eltis LD
Investigation of the role of a surface patch in the self-association of Chromatium vinosum high potential iron-sulfur protein.
Biochim Biophys Acta. 1999 Aug 17;1433(1-2):159-69., [PMID:10446369]

Abstract [show]
The role of a flattened, relatively hydrophobic surface patch in the self-association of Chromatium vinosum HiPIP was assessed by substituting phenylalanine 48 with lysine. The reduction potential of the F48K variant was 26 mV higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Nuclear magnetic resonance spectroscopy (NMR) revealed that the electronic structure of the oxidized cluster in these two proteins is very similar at 295 K. In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the introduction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Moreover, the substitution at position 48 abolished the fine structure in the g(z) region of the electron paramagnetic resonance (EPR) spectrum of oxidized C. vinosum rcHiPIP recorded in the presence of 1 M sodium chloride. These results support the hypothesis that the flattened, relatively hydrophobic patch mediates interaction between two molecules of HiPIP and that freezing-induced dimerization of the HiPIP mediated by this patch is responsible for the unusual fine structure observed in the EPR spectrum of the oxidized C. vinosum HiPIP.

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No. Sentence Comment
1 Grant Mauk c , Emmanuelle Boubour d , R. Bruce Lennox d , Lindsay D. Eltis aY * a Department of Biochemistry and the Centre de Recherche sur la Fonction, la Structure et l'Inge¨nierie des Prote¨ines, Pavillon Marchand, Universite¨ Laval, Quebec City, P.Q., Canada G1K 7P4 b Department of Chemistry and the Centre de Recherche sur la Fonction, la Structure et l'Inge¨nierie des Prote¨ines, Pavillon Vachon, Universite¨ Laval, Quebec City, P.Q., Canada G1K 7P4 c Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, B.C., Canada V6T 1Z3 d Department of Chemistry, McGill University, Montreal, P.Q., Canada H3A 2K6 Received 3 May 1999; received in revised form 8 June 1999; accepted 8 June 1999 Abstract The role of a flattened, relatively hydrophobic surface patch in the self-association of Chromatium vinosum HiPIP was assessed by substituting phenylalanine 48 with lysine. Login to comment
2 The reduction potential of the F48K variant was 26 mV higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Login to comment
4 In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the introduction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Login to comment
45 The nucleotide sequence of the gene encoding the F48K variant was veri'ed. Login to comment
96 Results and discussion Approximately 30 mg of puri'ed F48K variant rcHiPIP was obtained per liter of cell culture, which is comparable to the yield of WT rcHiPIP. Login to comment
97 Consistent with the introduction of an additional positive surface charge, F48K eluted from the anion-exchange column in a bu¡er containing 55 mM less NaCl than that required to elute WT rcHiPIP. Login to comment
99 The absorption spectra of WT and F48K rcHiPIPs were essentially identical (results not shown) and the R-factors (A280/A375) of the freshly oxidized WT and F48K variant rcHiPIPs were 2.18 and 2.13, respectively. Login to comment
104 300 MHz 1 H-NMR spectra of the oxidized WT (upper trace) and F48K (second trace) C. vinosum rcHiPIPs. Login to comment
106 The third and fourth traces represent the NOE spectra obtained from the selective irradiation of resonances A and B, respectively, of the F48K variant rcHiPIP. Login to comment
120 The midpoint reduction potentials of the WT and the F48K C. vinosum HiPIPs were 332 and 358 þ 5 mV versus SHE (20 mM MOPS, 80 mM NaCl, pH 7.0, 295 K), respectively. Login to comment
121 At a sweep rate of 10 mV/s, the separations between the anodic and cathodic current peaks were 62 mV and 78 mV for the WT and F48K variant HiPIPs, respectively. Login to comment
125 1 H-NMR spectra of the reduced C. vinosum WT and F48K rcHiPIPs were essentially identical, each containing four paramagnetically shifted signals (Table 1). Login to comment
127 The similarity of the chemical shifts of these resonances in F48K and WT rcHiPIP indicates that the Fe-S-C-H dihedral angles of the ligands are essentially identical in these two proteins and thus that the substitution at position 48 Fig. 3. Login to comment
132 1 H-NMR spectra of the oxidized C. vinosum WT and F48K rcHiPIPs were quite similar, each containing nine paramagnetically shifted signals (Fig. 2). Login to comment
134 The hyper'ne shifted resonances of the F48K variant were assigned by comparing their temperature dependences and NOEs to those of the WT HiPIP resonances. Login to comment
138 1D-NOE experiments were performed to assign resonances A, B, C, D and E of the F48K variant. Login to comment
140 The complete assignment of the hyper'ne shifted resonances of the oxidized C. vinosum F48K variant is summarized in Table 1. Login to comment
141 The 1 H-NMR spectrum of oxidized C. vinosum F48K rcHiPIP is very similar to that of the F48R variant [31]. Login to comment
143 The chemical shifts of the L-CH2 protons of Cys-46 and Cys-77 in the oxidized F48K variant indicate that the iron ligated to the former was slightly more ferric in character whereas that ligated to Cys-77 was slightly more mixed-valent in character, the shift being approximately 5 percentage points. Login to comment
149 EPR spectra of oxidized WT and F48K C. vinosum rcHiPIPs. Login to comment
153 These resonances had similar T1 in the WT and F48K variant rcHiPIPs, indicating that the observed di¡erences in magnetization exchange in these proteins did not arise from di¡erences in longitudinal relaxation rates (results not shown). Login to comment
159 In contrast to the magnetization transfer observed in WT C. vinosum rcHiPIP, no variation in the intensity of signals bP or cP was detected for the F48K variant upon saturation of signal A and B, at ionic strengths of 100 and 500 mM. Login to comment
161 The lower kex of the F48K variant with respect to the WT rcHiPIP supports the conclusion that the hydrophobic surface patch identi'ed by others [15^17] mediates interaction between two molecules of C. vinosum HiPIP in solution. Login to comment
165 The EPR spectrum of F48K recorded under similar conditions is essentially identi- Table 1 Summary of the 1 H NMR assignments of the hyper'ne shifted ligand proton signals of the WT and F48K C. vinosum rcHiPIP Residue Proton Oxidized Reduced Signal Chemical shift (ppm) Signal Chemical shift (ppm) WT F48K WT F48K Cys-43 HL1 H 335.5 (pC) 336.2 (pC) HL2 I 335.9 (pC) 336.2 (pC) aP 16.4 16.8 Cys-46 HL1 F 24.7 (aC) 18.4 (aC) HL2 G 24.4 (aC) 17.5 (aC) dP 10.0 11.3 Cys-63 HL1 C 36.8 (C) 37.4 (C) HL2 A 108.9 (C) 111.2 (C) bP 15.8 15.9 Cys-77 HL1 B 39.5 (C) 43.5 (C) cP 12.6 12.5 HL2 D 30.2 (C) 35.6 (C) HK E 27.4 (C) 29.2 (C) Values were determined in 5 mM sodium phosphate/D2O 99.95%, pH 7.0, 295 K. Login to comment
169 In contrast, the EPR spectrum of the F48K variant was una¡ected by the presence of 1 M NaCl (Fig. 4). Login to comment
171 The NMR and EPR studies of the F48K variant strongly support this hypothesis in two respects. Login to comment
172 First, the NMR spectrum of the oxidized F48K variant indicates that the substitution at position 48 only minimally a¡ects the electron distribution of the [Fe4S4]3 cluster at 295 K. Login to comment
173 It is thus unlikely that the di¡erences in the gz region of the `high salt' EPR spectra of F48K and WT rcHiPIPs arose from di¡erences in the electron distributions of their respective clusters at 10 K. Second, the lower kex of F48K with respect to that of WT HiPIP indicates that the introduction of a positive charge in the £attened, predominantly hydrophobic surface patch of the protein impedes self-association. Login to comment
176 Oxidized WT and F48K variant rcHiPIPs eluted from the same column equilibrated with 20 mM MOPS, 480 mM NaCl, pH 7.0 with an apparent molecular weight of 12.9 þ 0.5 kDa. Login to comment
179 This minor component, whose most prominent feature was a gy = 2.07, was present in the EPR spectra of F48K and WT C. vinosum rcHiPIP (Fig. 4) even though these preparations were of higher purity than the samples used in the previous study and were puri'ed from a di¡erent source. Login to comment