10 research outputs found
Identification of Unknown Protein Function Using Metabolite Cocktail Screening
SummaryProteins of unknown function comprise a significant fraction of sequenced genomes. Defining the roles of these proteins is vital to understanding cellular processes. Here, we describe a method to determine a protein function based on the identification of its natural ligand(s) by the crystallographic screening of the binding of a metabolite library, followed by a focused search in the metabolic space. The method was applied to two protein families with unknown function, PF01256 and YjeF_N. The PF01256 proteins, represented by YxkO from Bacillus subtilis and the C-terminal domain of Tm0922 from Thermotoga maritima, were shown to catalyze ADP/ATP-dependent NAD(P)H-hydrate dehydratation, a previously described orphan activity. The YjeF_N proteins, represented by mouse apolipoprotein A-I binding protein and the N-terminal domain of Tm0922, were found to interact with an adenosine diphosphoribose-related substrate and likely serve as ADP-ribosyltransferases. Crystallographic screening of metabolites serves as an efficient tool in functional analyses of uncharacterized proteins
Mapping of the immunodominant regions of the NAD-dependent formate dehydrogenase
AbstractA panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 has been obtained. The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test. The data obtained were interpreted residing on the structural model of the formate dehydrogenase at 3 Ă
resolution. The immunodominant regions in the formate dehydrogenase molecule and the epitopes for the monoclonal antibodies were elucidated
Substrate Specificity of Mammalian N-Terminal α-Amino Methyltransferase NRMT
N-Terminal methylation of free α-amino groups is
a post-translational
modification of proteins that was first described 30 years ago but
has been studied very little. In this modification, the initiating
M residue is cleaved and the exposed α-amino group is mono-,
di-, or trimethylated by NRMT, a recently identified N-terminal methyltransferase.
Currently, all known eukaryotic α-amino-methylated proteins
have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT
can also methylate artificial substrates in vitro in which X is G,
F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N-terminal
amino acids are variable with respect to the identity of X. Here we
use in vitro peptide methylation assays and substrate immunoprecipitations
to show that the canonical M-X-P-K methylation motif is not the only
one recognized by NRMT. We predict that N-terminal methylation is
a widespread post-translational modification and that there is interplay
between N-terminal acetylation and N-terminal methylation. We also
use isothermal calorimetry experiments to demonstrate that NRMT can
efficiently recognize and bind to its fully methylated products
Characterization of the Gating Brake in the I-II Loop of Cav3.2 T-type Ca2+ Channels*Sâ
Mutations in the I-II loop of Cav3.2 channels were discovered in
patients with childhood absence epilepsy. All of these mutations increased the
surface expression of the channel, whereas some mutations, and in particular
C456S, altered the biophysical properties of channels. Deletions around C456S
were found to produce channels that opened at even more negative potentials
than control, suggesting the presence of a gating brake that normally prevents
channel opening. The goal of the present study was to identify the minimal
sequence of this brake and to provide insights into its structure. A peptide
fragment of the I-II loop was purified from bacteria, and its structure was
analyzed by circular dichroism. These results indicated that the peptide had a
high α-helical content, as predicted from secondary structure
algorithms. Based on homology modeling, we hypothesized that the proximal
region of the I-II loop may form a helix-loop-helix structure. This model was
tested by mutagenesis followed by electrophysiological measurement of channel
gating. Mutations that disrupted the helices, or the loop region, had profound
effects on channel gating, shifting both steady state activation and
inactivation curves, as well as accelerating channel kinetics. Mutations
designed to preserve the helical structure had more modest effects. Taken
together, these studies showed that any mutations in the brake, including
C456S, disrupted the structural integrity of the brake and its function to
maintain these low voltage-activated channels closed at resting membrane
potentials