KChannelDB: Extraction of mutation data from the literature 2005, KChannelDB.
This data was extracted from Medline abstracts and full texts (when available) in an automated manner.
The table below describes the selected point mutation and provides links to other documents. The sentence(s) where the point mutation R264E was found are listed after the table.
The mutated residues are also indicated in the family sequence alignments and hyperlinked to the corresponding mutation pages.
Point mutation R264E in KCAB2_HUMAN
- The site-directed mutants R264E and N333W did not bind NADPH , whereas , the K d NADPH of Q214R was 10-fold greater than the wild-type protein
- The tetrameric structure of the wild-type protein was retained by the R264E mutant , indicating that NADPH binding is not a prerequisite for multimer formation
- The following primers were used: CTGGGGCACATCAATGTGGAGCTCCATGGAG (R189M) , GGTGCCATGACCGCGTCCCCTCTGGCGTGC (W243A) , GGTGCCATGACCTACTCCCCTCTGGCGTGC (W243Y) , CTGGTCCCCTCTGGCGTCCGGCATCGTC (C248S) , GTCTCAGGGAAGTTTGACAGCGGGATCCCAC (Y255F) , CCCATCTGCGAGCGAGCGGAATATCAC (Q214R) , CACCCTACTCCGAAGCCTCCCTGAAG (R264E) , CAACTTATGGAGTGGATTGGAGCAATACAG (N333W)
- The fluorescence of R264E was not quenched even by the addition of 1 mM of NADPH
- As expected , the freshly purified N333G and R264E proteins displayed a much stronger emission band at 335 nm than did equimolar concentrations of the WT or the C248S protein
- Both the WT and C248S proteins displayed an additional band at 450 nm , which was absent in the emission spectra of the N333W and the R264E proteins (Fig. 6 ) , indicating that the N333W and R264E proteins do not bind NADPH
- When excited at 340 nm (to elicit NADPH fluorescence) , both the WT and the C248S proteins displayed strong emission near 450 nm , whereas the N333W and the R264E proteins did not ; confirming that the N333W and R264E proteins do not contain NADPH bound to their active sites
- To examine whether the lack of NADPH binding affects the quaternary structure of the protein , we determined the Strokes radius of R264E using size exclusion chromatography
- The R264E protein eluted from the HPLC column with a retention time of 9.7 min (data not shown) , which was similar to the retention time of the WT protein , indicating that binding of NADPH is not essential for the formation of the Delta NK v beta 2 homotetramers
- The fluorescence spectra of the WT , C248S , R264E , and N333G Delta NK v beta 2
- This ionization may be due to Arg-264 , but the role of this residue could not be tested further because the R264E mutant did not bind NADPH
- The predominant role of Arg-264 in pyridine coenzyme binding to K v beta 2 is suggested by the observation that the R264E mutation prevented NADPH binding (K d > 1 mM)
- In contrast , the lack of coenzyme binding to the R264E mutant of K v beta 2 demonstrates that the same arginine residue binds to both the 2'-ribose phosphate and the pyrophosphate backbone of NADPH , as is evident from the crystal structure (12 )
- Our results showing that R264E , which does not bind NADPH , is a homotetramer suggest that nucleotide binding is not a prerequisite for maintaining structural integrity or for K v beta -K v beta interactions
- Specific mutations that can increase (C248S) , decrease (Q214E) , or prevent (R264E and N333W) NADPH binding to K v beta 2 were identified
F.Horn (kchanneldbcmbi.ru.nl), 17-Aug-2005