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The complex of cytochrome f and plastocyanin from Nostoc sp. PCC 7119 is highly dynamic.

Scanu S, Förster J, Finiguerra MG, Shabestari MH, Huber M, Ubbink M - Chembiochem (2012)

Bottom Line: The complex from Nostoc sp.Chemical-shift-perturbation analysis showed that the presence of spin labels on the surface of Cyt f does not significantly affect the binding of Pc.The paramagnetic relaxation enhancement results are not consistent with a single orientation of Pc, thus indicating that multiple orientations must occur and suggesting that an encounter state represents a large fraction of the complex.

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Affiliation: Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

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The interaction of Nostoc Zn-substituted Pc with wt Cytf and MTS-conjugated variants. A) Binding curvesfor selected residues were fitted globally to a 1:1 binding model [Eq.(2)]. B) Chemical shift perturbation maps of Zn-substituted Pc in thepresence of wild-type and MTS-conjugated Cyt f,color-coded on a surface model of Pc (PDB ID: 2CJ3). Red,Δδavg≥0.10 ppm;orange, Δδavg≥0.05 ppm;yellow, Δδavg≥0.02 ppm;blue, Δδavg<0.02 ppm.Prolines and residues with overlapping resonances are shown in gray.
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fig02: The interaction of Nostoc Zn-substituted Pc with wt Cytf and MTS-conjugated variants. A) Binding curvesfor selected residues were fitted globally to a 1:1 binding model [Eq.(2)]. B) Chemical shift perturbation maps of Zn-substituted Pc in thepresence of wild-type and MTS-conjugated Cyt f,color-coded on a surface model of Pc (PDB ID: 2CJ3). Red,Δδavg≥0.10 ppm;orange, Δδavg≥0.05 ppm;yellow, Δδavg≥0.02 ppm;blue, Δδavg<0.02 ppm.Prolines and residues with overlapping resonances are shown in gray.

Mentions: 15N-enriched Pc was produced in a cytoplasmic expression system (seethe Experimental Section) with a ZnII ion in the copper binding siteto eliminate the paramagnetic effect of CuII and possibleinterference from electron transfer reactions.[20] To establish whether the introduction of a probe interfereswith the Pc–Cyt f interaction, chemical shift analyseswere carried out for all variants. First, Zn-substituted [15N]Pc wastitrated with wild-type Cyt f, and HSQC spectra were acquiredat each titration point. The binding constant was obtained by fitting thechemical shift perturbation curves for the most affected amide groups (Figure 2 A), thereby yielding aKd of 8(3)×10−5m (estimated experimental error in parentheses), similar to thereported values of 3.8(0.1)×10−5m for Cu-Pc[21] and6.2(0.9)×10−5m for Cd-substituted Pc.[21]


The complex of cytochrome f and plastocyanin from Nostoc sp. PCC 7119 is highly dynamic.

Scanu S, Förster J, Finiguerra MG, Shabestari MH, Huber M, Ubbink M - Chembiochem (2012)

The interaction of Nostoc Zn-substituted Pc with wt Cytf and MTS-conjugated variants. A) Binding curvesfor selected residues were fitted globally to a 1:1 binding model [Eq.(2)]. B) Chemical shift perturbation maps of Zn-substituted Pc in thepresence of wild-type and MTS-conjugated Cyt f,color-coded on a surface model of Pc (PDB ID: 2CJ3). Red,Δδavg≥0.10 ppm;orange, Δδavg≥0.05 ppm;yellow, Δδavg≥0.02 ppm;blue, Δδavg<0.02 ppm.Prolines and residues with overlapping resonances are shown in gray.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3569876&req=5

fig02: The interaction of Nostoc Zn-substituted Pc with wt Cytf and MTS-conjugated variants. A) Binding curvesfor selected residues were fitted globally to a 1:1 binding model [Eq.(2)]. B) Chemical shift perturbation maps of Zn-substituted Pc in thepresence of wild-type and MTS-conjugated Cyt f,color-coded on a surface model of Pc (PDB ID: 2CJ3). Red,Δδavg≥0.10 ppm;orange, Δδavg≥0.05 ppm;yellow, Δδavg≥0.02 ppm;blue, Δδavg<0.02 ppm.Prolines and residues with overlapping resonances are shown in gray.
Mentions: 15N-enriched Pc was produced in a cytoplasmic expression system (seethe Experimental Section) with a ZnII ion in the copper binding siteto eliminate the paramagnetic effect of CuII and possibleinterference from electron transfer reactions.[20] To establish whether the introduction of a probe interfereswith the Pc–Cyt f interaction, chemical shift analyseswere carried out for all variants. First, Zn-substituted [15N]Pc wastitrated with wild-type Cyt f, and HSQC spectra were acquiredat each titration point. The binding constant was obtained by fitting thechemical shift perturbation curves for the most affected amide groups (Figure 2 A), thereby yielding aKd of 8(3)×10−5m (estimated experimental error in parentheses), similar to thereported values of 3.8(0.1)×10−5m for Cu-Pc[21] and6.2(0.9)×10−5m for Cd-substituted Pc.[21]

Bottom Line: The complex from Nostoc sp.Chemical-shift-perturbation analysis showed that the presence of spin labels on the surface of Cyt f does not significantly affect the binding of Pc.The paramagnetic relaxation enhancement results are not consistent with a single orientation of Pc, thus indicating that multiple orientations must occur and suggesting that an encounter state represents a large fraction of the complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

Show MeSH
Related in: MedlinePlus