An Active, Ligand-Responsive Pulling Geometry Reports on Internal Signaling between Subdomains of the DnaK Nucleotide-Binding Domain in Single-Molecule Mechanical Experiments

Abstract

Single-molecule mechanical experiments have proven to be ideal tools for probing the energetics and mechanics of large proteins and domains. In this paper, we investigate the nucleotide-dependent unfolding mechanics of the nucleotide-binding domain (NBD) of the Hsp70 chaperone DnaK. The NBD binds ADP or ATP in the binding cleft formed by lobe I and lobe II, which consists of two subdomains each. When force is applied to the termini of the NBD, the observed unfolding forces are independent of the nucleotide state. In contrast, when force is applied across the nucleotide-binding pocket, the unfolding forces report specifically on the nucleotide–phosphate state. In this active, ligand-responsive pulling geometry, we observed a bifurcation of the unfolding pathway; the pathway proceeds either through a cooperative “coupled pathway” or through a noncooperative “uncoupled pathway”. The partitioning between individual unfolding pathways can be effectively tuned by mutation or by the nucleotide exchange factor GrpE, i.e., by the factors affecting the strength of the lobe I–lobe II interactions within the native NBD. These experiments provide important insight into the molecular origin of the internal signaling between the subdomains of the nucleotide-binding domain of Hsp70 proteins and how signals are efficiently transferred inside the protein molecule.

Single‐Molecule FRET of Membrane Transport Proteins

“…For the last two decades, [1] smFRET techniques have been extensively used to study the properties of molecular machines, [2] intrinsically disordered proteins (IDPs),[ 3 , 4 , 5 , 6 , 7 , 8 ] protein folding processes,[ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ] protein‐ligand[ 17 , 18 , 19 ] and protein‐nucleic acid interactions,[ 20 , 21 , 22 , 23 ] as well as other structure‐function relationships and dynamic processes. [ 24 , 25 , 26 ] SmFRET is a particularly powerful and versatile tool to gain molecular and mechanistic insights because of its high spatial resolution (2–10 nm) combined with a wide range of accessible timescales (ns‐minutes). [ 1 , 10 , 27 ] In fact, smFRET is becoming increasingly popular in the investigation of membrane transporter dynamics (Table 1 ), thus complementing well‐established methods such as Nuclear Magnetic Resonance (NMR),[ 28 , 29 , 30 ] Small‐Angle X‐ray Scattering (SAXS)[ 31 , 32 , 33 ] and Electron Paramagnetic Resonance (EPR).…”

Classifying Residues in Mechanically Stable and Unstable Substructures Based on a Protein Sequence: The Case Study of the DnaK Hsp70 Chaperone

“…Hsp70 consists of 638 amino acid residues that belong to either mechanically stable or unstable protein substructures (Figure 1a)-based on our previous research [16,17,19,20]. For our analysis, we excluded residues 604-638 because they belong to a low-complexity intrinsically disorder sequence, which differs dramatically from the folded substructures.…”

“…Hence, some of the challenges are inherent to the cooperative nature of substructures and to the mechanical anisotropy of proteins, which means that the mechanical properties of proteins are highly dependent on pulling orientation [13][14][15][16]. Single-molecule studies showed that during the unfolding of large proteins, folded protein substructures are disrupted in distinct and well-defined steps [16][17][18][19][20][21][22][23][24][25]. The high reproducibility and specificity of these microscopic unfolding steps indicates a significant level of cooperativity.…”

“…Recently, we conducted a series of single-molecule nanomechanical studies on DnaK, the Hsp70 chaperone from E. coli [16,17,19,20]. Using laser optical tweezers for mechanical studies, we examined mechanical properties of both Hsp70 domains: the nucleotidebinding domain (NBD) and the substrate-binding domain (SBD).…”

Interpretation of Single-Molecule Force Experiments on Proteins Using Normal Mode Analysis

“…It is one of the most conserved proteins, with 40-60% identity between prokaryotic and eukaryotic homologues [43], and it plays key roles in protein folding, disaggregation, and degradation [44,45]. The E. coli homologue of the Hsp70 chaperone, DnaK, is probably the most thoroughly studied [46], and it has been extensively studied using optical trap experiments in our laboratory for a number of years [47][48][49][50]. Hsp70 can be divided into two major subunits, the nucleotide-binding domain (NBD) and the substrate-binding domain (SBD), both of which undergo conformational changes as a result of binding either the nucleotide or a substrate peptide.…”