Characterization of a highly purified, fully active, crystallizable RC-LH1-PufX core complex from Rhodobacter sphaeroides

E. C. Abresch; H. L.A. Axelrod; J. T. Beatty; J. A. Johnson; R. Nechushtai; M. L. Paddock

doi:10.1007/s11120-005-5106-z

Characterization of a highly purified, fully active, crystallizable RC-LH1-PufX core complex from Rhodobacter sphaeroides

E. C. Abresch, H. L.A. Axelrod, J. T. Beatty^*, J. A. Johnson, R. Nechushtai, M. L. Paddock

^*Corresponding author for this work

Department of Plant and Environmental Sciences (Natural Sciences)

Research output: Contribution to journal › Article › peer-review

26 Scopus citations

Abstract

Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1-PufX complex (RC-LH1-PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQ_A → D ⁺Q _A ^- ) in the RC as a function of light intensity. The RC-LH1-PufX complex had a quantum efficiency of 0.95 ± 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 ± 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 ± 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 Å. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 Å, b = 137.2 Å, c = 137.5 Å; α = 60.0°, β = 89.95°, γ =90.02°.

Original language	English
Pages (from-to)	61-70
Number of pages	10
Journal	Photosynthesis Research
Volume	86
Issue number	1-2
DOIs	https://doi.org/10.1007/s11120-005-5106-z
State	Published - Nov 2005

Bibliographical note

Funding Information:
We thank E. Li and D. Goodlett at the Institute for Systems Biology, Seattle, Washington, USA, and L. Gross at the University of California at San Diego for the mass spectroscopic identification of the bands on the SDS gel of the RC–LH1–PufX complex. We thank M. Okamura and G. Feher for helpful discussions. This work was supported by grants from the CIHR and NSERC (JTB).

Keywords

Bacterial photosynthesis
Core-complex
Light harvesting
PufX
RC-LH1
Reaction center

Access to Document

10.1007/s11120-005-5106-z

Cite this

@article{ddec529c36704346aea2c22f743e57fe,

title = "Characterization of a highly purified, fully active, crystallizable RC-LH1-PufX core complex from Rhodobacter sphaeroides",

abstract = "Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1-PufX complex (RC-LH1-PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQA → D +Q A - ) in the RC as a function of light intensity. The RC-LH1-PufX complex had a quantum efficiency of 0.95 ± 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 ± 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 ± 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 {\AA}. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 {\AA}, b = 137.2 {\AA}, c = 137.5 {\AA}; α = 60.0°, β = 89.95°, γ =90.02°.",

keywords = "Bacterial photosynthesis, Core-complex, Light harvesting, PufX, RC-LH1, Reaction center",

author = "Abresch, {E. C.} and Axelrod, {H. L.A.} and Beatty, {J. T.} and Johnson, {J. A.} and R. Nechushtai and Paddock, {M. L.}",

note = "Funding Information: We thank E. Li and D. Goodlett at the Institute for Systems Biology, Seattle, Washington, USA, and L. Gross at the University of California at San Diego for the mass spectroscopic identification of the bands on the SDS gel of the RC–LH1–PufX complex. We thank M. Okamura and G. Feher for helpful discussions. This work was supported by grants from the CIHR and NSERC (JTB).",

year = "2005",

month = nov,

doi = "10.1007/s11120-005-5106-z",

language = "אנגלית",

volume = "86",

pages = "61--70",

journal = "Photosynthesis Research",

issn = "0166-8595",

publisher = "Springer Netherlands",

number = "1-2",

}

TY - JOUR

T1 - Characterization of a highly purified, fully active, crystallizable RC-LH1-PufX core complex from Rhodobacter sphaeroides

AU - Abresch, E. C.

AU - Axelrod, H. L.A.

AU - Beatty, J. T.

AU - Johnson, J. A.

AU - Nechushtai, R.

AU - Paddock, M. L.

N1 - Funding Information: We thank E. Li and D. Goodlett at the Institute for Systems Biology, Seattle, Washington, USA, and L. Gross at the University of California at San Diego for the mass spectroscopic identification of the bands on the SDS gel of the RC–LH1–PufX complex. We thank M. Okamura and G. Feher for helpful discussions. This work was supported by grants from the CIHR and NSERC (JTB).

PY - 2005/11

Y1 - 2005/11

N2 - Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1-PufX complex (RC-LH1-PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQA → D +Q A - ) in the RC as a function of light intensity. The RC-LH1-PufX complex had a quantum efficiency of 0.95 ± 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 ± 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 ± 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 Å. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 Å, b = 137.2 Å, c = 137.5 Å; α = 60.0°, β = 89.95°, γ =90.02°.

AB - Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1-PufX complex (RC-LH1-PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQA → D +Q A - ) in the RC as a function of light intensity. The RC-LH1-PufX complex had a quantum efficiency of 0.95 ± 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 ± 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 ± 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 Å. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 Å, b = 137.2 Å, c = 137.5 Å; α = 60.0°, β = 89.95°, γ =90.02°.

KW - Bacterial photosynthesis

KW - Core-complex

KW - Light harvesting

KW - PufX

KW - RC-LH1

KW - Reaction center

UR - http://www.scopus.com/inward/record.url?scp=25444464841&partnerID=8YFLogxK

U2 - 10.1007/s11120-005-5106-z

DO - 10.1007/s11120-005-5106-z

M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???

C2 - 16172926

AN - SCOPUS:25444464841

SN - 0166-8595

VL - 86

SP - 61

EP - 70

JO - Photosynthesis Research

JF - Photosynthesis Research

IS - 1-2

ER -

Characterization of a highly purified, fully active, crystallizable RC-LH1-PufX core complex from Rhodobacter sphaeroides

Abstract

Bibliographical note

Keywords

Access to Document

Other files and links

Fingerprint

Cite this