Probing Small Molecule Binding to Unfolded Polyprotein Based on its Elasticity and Refolding

Abstract

Unfolded protein, a disordered structure found before folding of newly synthesized protein or after protein denaturation, is a substrate for binding by many cellular factors such as heat-stable proteins, chaperones, and many small molecules. However, it is challenging to directly probe such interactions in physiological solution conditions because proteins are largely in their folded state. In this work we probed small molecule binding to mechanically unfolded polyprotein using sodium dodecyl sulfate (SDS) as an example. The effect of binding is quantified based on changes in the elasticity and refolding of the unfolded polyprotein in the presence of SDS. We show that this single-molecule mechanical detection of binding to unfolded polyprotein can serve, to our knowledge, as a novel label-free assay with a great potential to study many factors that interact with unfolded protein domains, which underlie many important biological processes.

Mechanical responses of the mechanosensitive unstructured domains in cardiac titin

“…Both the PEVK and N2B-Us can be well fitted with the classic WLC model with a bending persistence length of 0.7-1.2 nm, which is consistent with the previously reported bending persistence of flexible polypeptide chain [Rief et al, 1999], and which was measured recently at low force [Winardhi et al, 2016].…”

Understanding the catch-bond kinetics of biomolecules on a one-dimensional energy landscape

“…AFM experiments show that at forces below 100 pN, titin I27 unfolding starts from the native state with the A-A′ strand stacked with both the B and G strands 27,28 . According to this hypothetical transition pathway suggested by the previous steered MD simulations and AFM experiments, δ l n à ¼ Lðn Ã Þ þ bðn Ã Þ À b 0 , where L(n * ) = n * × 0.38 nm is the contour length of the peeled peptide 29 , b(n * ) is the length connecting the n * + 1 residue and the C-terminus of I27 and b 0~4 .32 nm is the length between N-and C-termini of I27 in its native state (Fig. 4a).…”

“…Thus, δ l n à of I27 can be calculated ( . The extension change at each testing value of n*, δ z ðF; δ l n à Þ, is calculated based on the known force-extension curves of a freely rotating rigid structure (Supplementary Figure 5) and a flexible peptide polymer with a certain bending persistence of A ∈ (0.5,1) nm 5,[29][30][31] ( Supplementary Note 4), which is also treated as a fitting parameter. At each value n * , the I27 experimental data 2 were fitted using Eq.…”

Structural-Elastic Determination of the Lifetime of Biomolecules Under Force

“…The typical sizes of protein domain and the folded core in the transition state are in the order of a few nanometers; therefore, k B T /b 0 and k B T /b * are close to 1 pN. If in the transition state a protein peptide or a ssDNA/ssRNA polymer is produced, due to their very small bending persistence of A ∼ 1 nm [28,34], k B T /A ∼ 5 pN becomes the predominating factor that imposes a restriction to the lower boundary of force range to apply Eq. 5.…”

“…x L * (F ) can be described by the worm-like chain (WLC) polymer model that contains two parameters, the bending persistence length A and the contour length L * = n * l r . In previous single-molecule manipulation experiments, values of A ∼ 0.8 nm and l r ∼ 0.38 nm for peptide chain have been experimentally determined [28]. Based on the WLC model, x L * (F ) can be analytically approximated by the Marko-Siggia formula [29]:…”