Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody.

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.

Predict prokaryotic proteins through detecting N-formylmethionine residues in protein sequences using support vector machine.

Identifying prokaryotes in silico is commonly based on DNA sequences. In experiments where DNA sequences may not be immediately available, we need to have a different approach to detect prokaryotes based on RNA or protein sequences. N-formylmethionine (fMet) is known as a typical characteristic of prokaryotes. A web tool has been implemented here for predicting prokaryotes through detecting the N-formylmethionine residues in protein sequences. The predictor is constructed using support vector machine. An online predictor has been implemented using Python. The implemented predictor is able to achieve the total prediction accuracy 80% with the specificity 80% and the sensitivity 81%.

Identification of retained N-formylmethionine in bacterial recombinant mammalian cytochrome P450 proteins with the N-terminal sequence MALLLAVFL...: roles of residues 3-5 in retention and membrane topology.

An N-terminal block to Edman degradation was observed when any of five different mammalian cytochrome P450 (P450) proteins was expressed in Escherichia coli using the N-terminal sequence MALLLAVFL... This block was also seen in Salmonella typhimurium. With all proteins examined, the block could be removed by mild acid hydrolysis (0.6--6 N HCl, 23 degrees C) to expose Met as the N-terminus, suggesting N-formylMet retention. The N-terminal peptide of a modified P450 1A2 ("mutant 1", containing a thrombin-sensitive site inserted at residue 25) was released with thrombin and analyzed by electrospray mass spectrometry and found to yield the M(r) expected for the N-formyl derivative (+/- 0.8 amu). The region of positions 3--5 was altered by random mutagenesis, and three P450 1A2-expressing clones were analyzed for nucleotide and amino acid sequences. The changes from LLL were to RER (P450 1A2a), VDS (P450 1A2b), and WRH (P450 1A2c); these all show slightly dissimilar hydropathy plots compared to the MALLLAVFL... sequence. Mutant P450 1A2a had the N-terminal Met removed to yield N-terminal Ala; P450 1A2b contained an unmodified Met at the N-terminus; P450 1A2c had an approximately 80% block of the N-terminal Met. Experiments with bacterial membranes containing expressed P450 1A2 mutant 1 and P450 1A2 mutant 2 (thrombin-sensitive site inserted at residue 46) suggest that thrombin site 2, but not 1, is sequestered in the membrane. Spheroplasts of bacteria expressing P450 1A2 and the mutants at positions 3--5 were treated with proteinase K; amino acid analysis indicated that no cleavage occurred. These results are interpreted in a model in which most of the mammalian P450 expressed in the bacterium is located in the cytosol, the region near residue 46 is in the inner membrane, the region near residue 25 is in the cytosol, and the N-terminus is either imbedded in the membrane or free in the cytosolic space, depending upon the sequence. However, the possibility that the differences in N-terminal processing are the result of direct changes in interactions with the deformylase and Met aminopeptidase cannot be excluded.