Information Technology Reference
In-Depth Information
signal peptidases (Lsp), cleave lipoproteins when a large hydrophobic residue is present at
the -3 position and a modified cysteine is present at the +1 position, being the consensus
cleavage site “A|(G/A)|C” [5].
The most currently used method to identify the presence and location of signal
peptides cleavage sites, in amino acid sequences from different organisms, is the neural
network-based signalP predictor. Signal IP combines two different neural networks, one
that discriminates between residues that belong and do not belong to a signal peptide (S-
core) and, one that was conceived to recognise signal peptidase cleavages sites (C-score)
[1]. The cleavage site is predicted by multiplying together the C-score and the negative
“derivative” of the S-score, while the discrimination between proteins that have and do not
have a signal peptide is based on the mean S-score evaluated from the N-terminus to the
predicted cleavage site.
SignalP V2.0 comprises two signal peptide prediction methods, Signal-NN (based
on neural networks) and, SignalP-HMM (based on hidden Markov models). According to
SignalP server (http://www.cbs.dtu.dk/services/SignalP-2.0) SignalP-H provides not only a
prediction of the presence of a signal peptide and the position of cleavage site, but also an
approximate assignment of n-, h- and c-regions within the signal peptide. Additionally, for
eukaryotic data, the HMM version has an improved discrimination between signal peptides
and uncleaved signal anchors, but has a lower accuracy in predicting the precise location of
the cleavage site [1].
Some proteins have sequences that initiate translocation in the same way as SPs do,
but are not cleaved by signal peptidase. As the rest of the polypeptide chain is translocated
trough the membrane, the resulting protein remains anchored to the membrane by the
hydrophobic region, with a short N-terminal cytoplasmic domain. The uncleaved signal
peptide is kwon as a signal anchor SA, and the resulting protein is known as a type II
membrane protein. SAs differ from SPs in other respects than the cleavage sites: they have
longer hydrophobic starches and the N-terminal region of the hydrophobic stretches can
also be much longer [6].
Multiheme cytocrome c nitrite reductase (ccNiR) isolated from the sulphate-
reducing bacteria Desulfovibrio desulfuricans ATCC 27774 is a membrane bound enzyme
that catalyses the dissimilatory nitrite reduction to ammonia in a six-electron step. It is a
key enzyme involved in the second and terminal step of the dissimilatory nitrate reduction
pathway of the nitrogen cycle and plays an important role on bacterial respiratory energy
conservation [7,8]. It has recently been shown that ccNiR is a hetero-oligomeric complex
composed by two subunits (63 KDa and 19 KDa) both containing c-type hemes, encoded
by genes nrfA and nrfA, respectively [9].
Based on the primary sequence determined by chemical and DNA sequencing
(described on references 9 and 10) we used a combined method of SignalP V2.0 (SignalP-
HMM and Signal-NN) in association with TMHMM 2.0 for the assessment of ccNir most
relevant topological characteristics.
1. Primary Structure
Chemical Sequencing. The N-terminal amino-acid sequence of D. desulfuricans
ATCC 27774 ccNiR subunits and their internal peptides were determined by automated
Edman degradation on a Procise¥ Protein Sequencer (model 491, Applied Biosystem) as
described, in detail, in the literature [9].
The internal peptide sequences obtained by enzymatic cleavage, as well as the nrfA
and nrfH sequences have been submitted to the EMBL database under the accession
