Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/201147
Type: Artigo de periódico
Title: Structural And Functional Characterization Of The Chaperone Hsp70 From Sugarcane. Insights Into Conformational Changes During Cycling From Cross-linking/mass Spectrometry Assays.
Author: Tiroli-Cepeda, Ana O
Lima, Tatiani B
Balbuena, Tiago S
Gozzo, Fábio C
Ramos, Carlos H I
Abstract: Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound. Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics.
Subject: Amino Acid Sequence
Binding Sites
Computer Simulation
Enzyme Activation
Hsp70 Heat-shock Proteins
Mass Spectrometry
Models, Chemical
Models, Molecular
Molecular Sequence Data
Protein Binding
Protein Conformation
Saccharum
Heat Shock Protein
Hsp70
Molecular Chaperone
Protein Dynamics
Protein Folding
Sugarcane
Citation: Journal Of Proteomics. v. 104, p. 48-56, 2014-Jun.
Rights: fechado
Identifier DOI: 10.1016/j.jprot.2014.02.004
Address: http://www.ncbi.nlm.nih.gov/pubmed/24530624
Date Issue: 2014
Appears in Collections:Unicamp - Artigos e Outros Documentos

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