7,939 research outputs found
Water structuring and collagen adsorption at hydrophilic and hydrophobic silicon surfaces
The adsorption of a collagen fragment on both a hydrophobic,
hydrogen-terminated and a hydrophilic, natively oxidised Si surface is
investigated using all-atom molecular dynamics. While favourable direct
protein-surface interactions via localised contact points characterise adhesion
to the hydrophilic surface, evenly spread surface/molecule contacts and
stabilisation of the helical structure occurs upon adsorption on the
hydrophobic surface. In the latter case, we find that adhesion is accompanied
by a mutual fit between the hydrophilic/hydrophobic pattern within the protein
and the layered water structure at the solid/liquid interface, which may
provide an additional driving force to the classic hydrophobic effect
Efficient unfolding pattern recognition in single molecule force spectroscopy data
BackgroundSingle-molecule force spectroscopy (SMFS) is a technique that measures the force necessary to unfold a protein. SMFS experiments generate Force-Distance (F-D) curves. A statistical analysis of a set of F-D curves reveals different unfolding pathways. Information on protein structure, conformation, functional states, and inter- and intra-molecular interactions can be derived.ResultsIn the present work, we propose a pattern recognition algorithm and apply our algorithm to datasets from SMFS experiments on the membrane protein bacterioRhodopsin (bR). We discuss the unfolding pathways found in bR, which are characterised by main peaks and side peaks. A main peak is the result of the pairwise unfolding of the transmembrane helices. In contrast, a side peak is an unfolding event in the alpha-helix or other secondary structural element. The algorithm is capable of detecting side peaks along with main peaks.Therefore, we can detect the individual unfolding pathway as the sequence of events labeled with their occurrences and co-occurrences special to bR\u27s unfolding pathway. We find that side peaks do not co-occur with one another in curves as frequently as main peaks do, which may imply a synergistic effect occurring between helices. While main peaks co-occur as pairs in at least 50% of curves, the side peaks co-occur with one another in less than 10% of curves. Moreover, the algorithm runtime scales well as the dataset size increases.ConclusionsOur algorithm satisfies the requirements of an automated methodology that combines high accuracy with efficiency in analyzing SMFS datasets. The algorithm tackles the force spectroscopy analysis bottleneck leading to more consistent and reproducible results
Structural interrogation of phosphoproteome identified by mass spectrometry reveals allowed and disallowed regions of phosphoconformation
High-throughput mass spectrometric (HT-MS) study is the method of choice for monitoring global changes in proteome. Data derived from these studies are meant for further validation and experimentation to discover novel biological insights. Here we evaluate use of relative solvent accessible surface area (rSASA) and DEPTH as indices to assess experimentally determined phosphorylation events deposited in PhosphoSitePlus. Based on accessibility, we map these identifications on allowed (accessible) or disallowed (inaccessible) regions of phosphoconformation. Surprisingly a striking number of HT- MS/MS derived events (1461/5947 sites or 24.6%) are present in the disallowed region of conformation. By considering protein dynamics, autophosphorylation events and/or the sequence specificity of kinases, 13.8% of these phosphosites can be moved to the allowed region of conformation. We also demonstrate that rSASA values can be used to increase the confidence of identification of phosphorylation sites within an ambiguous MS dataset. While MS is a stand-alone technique for the identification of vast majority of phosphorylation events, identifications within disallowed region of conformation will benefit from techniques that independently probe for phosphorylation and protein dynamics. Our studies also imply that trapping alternate protein conformations may be a viable alternative to the design of inhibitors against mutation prone drug resistance kinases
Universality and diversity of folding mechanics for three-helix bundle proteins
In this study we evaluate, at full atomic detail, the folding processes of
two small helical proteins, the B domain of protein A and the Villin headpiece.
Folding kinetics are studied by performing a large number of ab initio Monte
Carlo folding simulations using a single transferable all-atom potential. Using
these trajectories, we examine the relaxation behavior, secondary structure
formation, and transition-state ensembles (TSEs) of the two proteins and
compare our results with experimental data and previous computational studies.
To obtain a detailed structural information on the folding dynamics viewed as
an ensemble process, we perform a clustering analysis procedure based on graph
theory. Moreover, rigorous pfold analysis is used to obtain representative
samples of the TSEs and a good quantitative agreement between experimental and
simulated Fi-values is obtained for protein A. Fi-values for Villin are also
obtained and left as predictions to be tested by future experiments. Our
analysis shows that two-helix hairpin is a common partially stable structural
motif that gets formed prior to entering the TSE in the studied proteins. These
results together with our earlier study of Engrailed Homeodomain and recent
experimental studies provide a comprehensive, atomic-level picture of folding
mechanics of three-helix bundle proteins.Comment: PNAS, in press, revised versio
Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations
Mutations create the genetic diversity on which selective pressures can act,
yet also create structural instability in proteins. How, then, is it possible
for organisms to ameliorate mutation-induced perturbations of protein stability
while maintaining biological fitness and gaining a selective advantage? Here we
used a new technique of site-specific chromosomal mutagenesis to introduce a
selected set of mostly destabilizing mutations into folA - an essential
chromosomal gene of E. coli encoding dihydrofolate reductase (DHFR) - to
determine how changes in protein stability, activity and abundance affect
fitness. In total, 27 E.coli strains carrying mutant DHFR were created. We
found no significant correlation between protein stability and its catalytic
activity nor between catalytic activity and fitness in a limited range of
variation of catalytic activity observed in mutants. The stability of these
mutants is strongly correlated with their intracellular abundance; suggesting
that protein homeostatic machinery plays an active role in maintaining
intracellular concentrations of proteins. Fitness also shows a significant
correlation with intracellular abundance of soluble DHFR in cells growing at
30oC. At 42oC, on the other hand, the picture was mixed, yet remarkable: a few
strains carrying mutant DHFR proteins aggregated rendering them nonviable, but,
intriguingly, the majority exhibited fitness higher than wild type. We found
that mutational destabilization of DHFR proteins in E. coli is counterbalanced
at 42oC by their soluble oligomerization, thereby restoring structural
stability and protecting against aggregation
Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP
Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post- translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B’. Results also show that unstructured post- ranslationally modified C-terminal tails are
responsible for the dynamics of Sm-B/B’ and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.This work was funded by: BBSRC (OVM), BBSRC and EPSRC (HH and NM), EU Prospects (HH), European Science Foundation (NM), the Royal Society (CVR), and fellowship from JSPS and HFSP (YM and DAPK respectively)
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