Hi all, late to the discussion as usual... Related to the issue of "why does my single point mutant appear to have residual activity", what do people here make of this warning from 1989? http://pubs.acs.org/doi/pdf/10.1021/ar00163a001 Some highlights:
"Substitution of the Ser68 codon with a codon for glycine (Gly) yields a beta-lactamase which has a low but reproducible activity that is about 0.1% of that of the wild-type enzyme" "The underlying assumption in all experiments of the sort described above is that knowledge of the nucleotide sequence of the gene for a protein is tantamount to knowledge of the amino acid sequence. There is ample evidence to support this assumption. However, there is also much evidence that the biochemical machinery which converts DNA sequences into proteins makes occasional mistakes. These can be errors in transcription and in translation, although the latter are probably more common. These mistakes can lead to microscopic heterogeneity of the proteins which are produced from a fixed DNA sequence, with the protein that is specified by the gene mixed with minute amounts of sequence variants. A variant produced by an error of translation, for example, can have an intrinsic activity that is different from that of the predominant species. This is the likely explanation for the putative activity of a beta-lactamase Gly68 mutant which turns out to have no measurable activity whatsoever." "Because of the degeneracy of the genetic code, most amino acids have more than one codon. The glycine codons are GCX, where X is A, G, C, or U. When the Ser68 AGC codon is changed to GGC, a low beta-lactamase activity is observed, as described above. When AGC is replaced instead by GGA, however, the mutant beta-lactamase is made in the same amount, but it has no detectable activity. Thus, the Gly68 mutant protein per se has no inherent activity. Instead, activity is correlated with the presence in the gene of a specific codon for glycine. The probable explanation is that serine is occasionally inserted instead of glycine at the GGC codon (at position 68) of the messenger RNA that is transcribed from the mutant gene." I don't think that is the case here, as there is no obvious path from Ala codons back to Cys codons (although Ser or Thr would be possible). This is meant as a more general question of particular interest in crystallography as we like to co-crystallize our enzymes in tiny solutions at several mg/ml for several weeks, which is akin to asking 0.1% activities to show their ugly heads. Cheers, Jose. ================================ Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dk<mailto:josea.cuesta.se...@carlsberglab.dk> ================================ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Monday, April 20, 2015 8:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cleaved peptide density! Dear Crystallographers, I am working with a cysteine protease. I co-crystallized the protease with some small chemically synthesised peptides of 7 amino acid residues. I mutated the active Cysteine residue with Alanine to avoid the peptide cleavage so that I can get the whole peptide bound with my protein. But interesting I got the density for a cleaved peptide with 4 amino acids instead of the whole peptide. The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred exactly at the same peptide bond where it should. But I do not know how! Now my question is why I am getting the cleaved peptide as I already mutated the active residue Cysteine with Alanine (this mutant did no show any activity when I checked with SDS-PAGE). If anybody has the same kind of experience please advice me. Thanks in advance. Best, Dipankar -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
