Abstract
Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be.
Highlights
Hsp70 molecular chaperones are part of the quality control machinery that functions to assist protein folding
Members of the Hsp70 family have been implicated in refolding of misfolded proteins, folding of newly synthesized polypeptide chains, disassembly of larger aggregates and translocation of proteins in organelles [1]
Hsp70 molecules enable cell survival during stress or heat-shock conditions that are characterized by an increased concentration of denatured polypeptides
Summary
Hsp molecular chaperones are part of the quality control machinery that functions to assist protein folding. Hsp molecules enable cell survival during stress or heat-shock conditions that are characterized by an increased concentration of (partially) denatured polypeptides. These chaperones recognize and bind misfolded or aggregationprone peptide stretches through exposed hydrophobic regions which are normally buried in the protein core. With ATP bound, substrate affinity is low and Hsp resides in an open state, ready to receive a suitable substrate. Once the substrate is bound, ATP is hydrolyzed to ADP and Hsp undergoes a conformational change to a high affinity state, subsequently trapping the substrate. Upon exchange of ADP for ATP, Hsp returns to a low affinity state, enabling binding of another substrate or providing another refolding cycle for the same substrate if necessary
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