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      Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain

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          Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release.

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          Most cited references 57

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          Calculation of protein extinction coefficients from amino acid sequence data.

          Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.
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            The leucine-rich repeat as a protein recognition motif.

             B Kobe (2001)
            Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna1p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.
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              The leucine-rich repeat: a versatile binding motif.

              Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. All proteins containing these repeats are thought to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape. These two features may be responsible for the protein-binding functions of proteins containing leucine-rich repeats.

                Author and article information

                [1 ]Department of Chemistry, University of Cambridge, Cambridge, United Kingdom
                [2 ]Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
                [3 ]Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, United Kingdom
                [4 ]CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (Consejo Superior de Investigaciones Científicas-University of Seville-UPO-Junta de Andalucia), Seville, Spain
                [5 ]Joint BSC-IRB Research Programme in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain
                [6 ]Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
                Semmelweis University, Hungary
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: FAA AD FS CR JLPB CMD NC. Performed the experiments: FAA AD FS JLPB. Analyzed the data: FAA AD FS CR JLPB PT XS CMD NC. Contributed reagents/materials/analysis tools: CVR CMD. Wrote the manuscript: FAA AD FS CR JLPB PT CVR XS CMD NC.


                Current address: Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                28 June 2013
                : 8
                : 6

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                FAA was recipient of a graduate fellowship from the Italian Ministry of Education, University and Research. AD is grateful for support from Murray Edwards College, Cambridge, through a Junior Research Fellowship. FS is a Sir Henry Wellcome Fellow. CR acknowledges financial support by the Spanish Ministry of Health according to the 'Plan Nacional de I+D+I 2008-2011', through ISCIII with cofunding by FEDER (CP10/00527). JLPB is a Royal Society University Research Fellow. FAA and PT are grateful for support from Regione Lombardia (NEDD and "Network Tecnologico integrato per lo studio proteomico e trascrittomico di malattie neurodegenerative correlate a deposizioni di amiloidi"). CMD acknowledges support from BBSRC (BB/E019927/1), the Wellcome Trust (094425/Z/10/Z), the European Commission (project LSHM-CT-2006-037525). NC acknowledges support from Human Frontiers Science Program (HFSP) through a Long-term Fellowship (LT000795/2009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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