Dissecting Cooperative Communications in a Protein with a High‐Throughput Single‐Molecule Scalpel

Abstract

Miscued communication often leads to misfolding and aggregation of the proteins involved in many diseases. Owing to the ensemble average property of conventional techniques, detailed communication diagrams are difficult to obtain. Mechanical unfolding affords an unprecedented perspective on cooperative transitions by observing a protein along a trajectory defined by two mutated cysteine residues. Nevertheless, this approach requires tedious sample preparation at the risk of altering native protein conformations. To address these issues, we applied click chemistry to tether a protein to the two dsDNA handles through primary amines in lysine residues as well as at the N terminus. As a proof of concept, we used laser tweezers to mechanically unfold and refold calmodulin along 36 trajectories, maximally allowed by this strategy in a single batch of protein preparation. Without a priori knowledge of the particular residues to which the double-stranded DNA handles attach, we used hierarchical cluster analysis to identify 20 major trajectories, according to the size and the pattern of unfolding transitions. We dissected the cooperativity into all-or-none and partially cooperative events, which represent strong and weak high-order interactions in proteins, respectively. Although the overall cooperativity is higher within the N or C lobe than that between the lobes, the all-or-none cooperativity is anisotropic among different the unfolding trajectories and becomes relatively more predominant when the size of the protein segments increases. The average cooperativity for all-or-none transitions falls within the expected range observed by ensemble techniques, which supports the hypothesis that unfolding of a free protein can be reconstituted from individual trajectories.

Exploded view of higher order G-quadruplex structures through click-chemistry assisted single-molecule mechanical unfolding

“…Detailed analysis revealed that 11% of all unfolding events were direct unfolding from fully folded structure (a*) to fully unfolded population (d). Such cooperative unfolding confirmed the presence of the higher order interaction between the two hTERT G-quadruplexes ( 26 ). For the rest of the molecules, majority (65%) preferred to unfold through the G-quadruplex formed in the 1–4 G-tracts, followed by the unfolding of the hTERT 5–12 G-quadruplex.…”

“…For example, in the 5′-end G-quadruplex (NMS1–4), the cooperative unfolding constituted 30% of all events (Figure 4A ) whereas it was 31% in the 3′-end G-quadruplex (NMS5–12, Figure 3 ). Previously, these cooperative unfolding events have been ascribed to the high-order interactions in proteins ( 26 ).…”

Single-Molecule Mechanics in Ligand Concentration Gradient

“…on proteins. Although the small size of usual proteins may pose experimental challenge, large filamentous proteins (e.g., titin [50]) or proteins captured with DNA handles [51][52][53][54] may be investigated directly. Considering the emerging significance of single-molecule mechanics in understanding structural and functional detail, the addition of precisely adjusted ligand concentration gradients, as demonstrated here, may provide further access to understanding the exact mechanisms behind biomolecular phenomena.…”

Interaction of G-Quadruplexes in the Full-Length 3′ Human Telomeric Overhang

“…To differentiate the higherorder G-quadruplex structures from the noninteracting G-quadruplexes in the 24G DNA construct, we introduced the hierarchical cluster assay (HCA). 36 Different from other discriminant assay such as Linear Discriminant Analysis (LDA, see below), the HCA does not require training to categorize groups in a sample. 37 Therefore, it is ideal to classify samples into various groups without prior knowledge of their identities.…”