ABCMdb

PMID: 10446369

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., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys The nucleotide sequence of the gene encoding the F48K variant was veri'ed. Login to comment 96 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 300 MHz 1 H-NMR spectra of the oxidized WT (upper trace) and F48K (second trace) C. vinosum rcHiPIPs. Login to comment 106 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 124 ABCC8 p.Phe48Arg For example, the reduction potentials of the F48R variant C. vinosum HiPIP was 23 mV higher than that of the WT [31] and that of the V66K variant Ectothiorhodospira halophila HiPIP was 29 mV higher than that of the WT [32]. Login to comment 125 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 1D-NOE experiments were performed to assign resonances A, B, C, D and E of the F48K variant. Login to comment 140 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys ABCC8 p.Phe48Arg 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys EPR spectra of oxidized WT and F48K C. vinosum rcHiPIPs. Login to comment 153 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 162 ABCC8 p.Phe48Arg Interestingly, the electron transfer self-exchange of F48R variant C. vinosum HiPIP has been calculated by EXSY to be one third of the kex calculated for the WT [31]. Login to comment 165 ABCC8 p.Phe48Lys ABCC8 p.Phe48Lys ABCC8 p.Phe48Lys ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys The NMR and EPR studies of the F48K variant strongly support this hypothesis in two respects. Login to comment 172 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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 ABCC8 p.Phe48Lys 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