Addition of C-terminal histidyl tags to PsaL and PsaK1 proteins of cyanobacterial photosystem I

Citation
Hd. Tang et Pr. Chitnis, Addition of C-terminal histidyl tags to PsaL and PsaK1 proteins of cyanobacterial photosystem I, I J BIOCH B, 37(6), 2000, pp. 433-440
Citations number
28
Categorie Soggetti
Biochemistry & Biophysics
Journal title
INDIAN JOURNAL OF BIOCHEMISTRY & BIOPHYSICS
ISSN journal
03011208 → ACNP
Volume
37
Issue
6
Year of publication
2000
Pages
433 - 440
Database
ISI
SICI code
0301-1208(200012)37:6<433:AOCHTT>2.0.ZU;2-S
Abstract
In vitro mutagenesis was used to produce two photosystem I mutants of the c yanobacterium Synechocystis sp. PCC 6803. The mutants HK and HL contained h exahistidyl tags at the C-termini of the PsaK1 and PsaL subunits, respectiv ely. The HK mutant contained wild-type amounts of trimeric PS I complexes, but the level of hexahistidine-tagged PsaK1 was found only ten per cent in the PS I complexes and membranes of the wild type level. Therefore, attachm ent of a tag at the C-terminus interferes with the expression or assembly o f PsaK1. In contrast, the HL mutant contained a similar level of tagged Psa L as that in the wild type. However, trimeric PS I complexes could not be o btained from this strain, indicating that the C-terminus of PsaL is involve d in the formation of PS I trimers. Hexahistidine-tagged complexes of the H L and HK strains could not be purified with Nickel-affinity chromatography, unless photosystem I was denatured with urea, demonstrating that tagged C- termini of PsaK1 and PsaL were embedded inside of the PS I complex. Protect ion of the C-terminus from trypsin cleavage further supported this conclusi on. Thus, histidine tagging allowed us to demonstrate role of C-termini of two proteins of photosystem I.