Probing the arrangement of the conformational ensembles of DnaK

Proteins are macromolecules that are important for life. Proteins have various cellular functions, which rely on their correctly folded three-dimensional (3-D) structure. Proper folding of nascent protein chains that require assistance to get their native state is accomplished by Heat shock proteins (Hsps) also called as "molecular chaperons". Hsps are highly conserved family of proteins from bacteria to human. They are not only expressed in normal conditions, but also are expressed in different stress conditions. They participate in numerous cellular functions, which include preventing aggregation, assisting protein folding, refolding of denatured proteins, and degradation. Hsp70s can recognize unfolded, misfolded and aggregated forms of the proteins from their exposed hydrophobic groove. There is a cycle of HSP70s sustained by the use of ATP. During this cycle client polypeptides are get folded by HSP70s successfully to their native states. Hsp70 proteins have two main domains, which are a highly conserved 44 kDa, N-terminal nucleotide binding domain (NBD) and a variable 25 kDa, C-terminal substrate binding domain (SBD). NBD comprises two main lobes and these lobs are divided into upper-lower part and right-left part. SBD consist of two parts which are α-helical lid and β-sandwich subdomain. The α-helical lid covers over the polypeptide bound state. The β-sandwich binds to substrates with a hydrophobic groove. NBD and SBD are connected to each other with a highly conserved hydrophobic linker (GDVKDVLLL). Hydrophobic linker has a critical role in allosteric mechanism of Hsp70. Two different constructs of DnaK namely, DnaK388 and DnaK392, are used to investigate the interdomain communication mechanism by Swain et al. (2007). Both residues do not contain SBD. DnaK392 contains a conserved hydrophobic 389VLLL392 sequence of the interdomain linker, while DnaK388 lacks this linker sequence. These investigations were shown with truncated DnaK constructs as presence or absence of linker. In the same study, the partial interdomain 389VLLL392 linker sequence is found as active and critical for the communication of the ATPase domain and the SBD. It is shown that the construct DnaK392 mimics very similar ATPase activity at the peptide bound state. It was reported that DnaK392 has an ATPase activity that is similar to that of the substrate-stimulated form of DnaK. On the contrary, DnaK388 has an ATPase activity that is similar to that of the unstimulated state of DnaK. In this study, we would like to detect the different adaptable conformations of the ATPase domain derived from the crucial 389VLLL392 linker in equilibrium. We studied with an NBD variant which has a partial interdomain linker sequence 388VLLL392 and with the NDB itself to investigate the arrangement of the conformational ensembles of Hsp70. These proteins are shown to be already folded after purification and they are found in APA state. It is shown by the native mass spectrometry charge state distribution. Our findings revealed that DnaK392 has a charge shift behaviour observed at +14 charge state rather than the most effective charge of +13 charge state after pH 7.5 by native mass spectrometry. Below pH 7.5 there is predominantly one population being observed; and with the pH increase, two distinct conformations of DnaK392 were determined by ion-mobility mass spectrometry. pH alterations cause subtle conformational variations for DnaK388, on the other hand, our complementary approach has shown the existence of a discrete allosterically active state of DnaK in the presence of partial linker using ion-mobility mass spectrometry.