Structural distributions from single-molecule measurements as a tool for molecular mechanics

Abstract

A mechanical view provides an attractive alternative for predicting the behavior of complex systems since it circumvents the resource-intensive requirements of atomistic models; however, it remains extremely challenging to characterize the mechanical responses of a system at the molecular level. Here, the structural distribution is proposed to be an effective means to extracting the molecular mechanical properties. End-to-end distance distributions for a series of short poly-L-proline peptides with the sequence PnCG3K-biotin (n = 8, 12, 15 and 24) were used to experimentally illustrate this new approach. High-resolution single-molecule Förster-type resonance energy transfer (FRET) experiments were carried out and the conformation-resolving power was characterized and discussed in the context of the conventional constant-time binning procedure for FRET data analysis. It was shown that the commonly adopted theoretical polymer models—including the worm-like chain, the freely jointed chain, and the self-avoiding chain—could not be distinguished by the averaged end-to-end distances, but could be ruled out using the molecular details gained by conformational distribution analysis because similar polymers of different sizes could respond to external forces differently. Specifically, by fitting the molecular conformational distribution to a semi-flexible polymer model, the effective persistence lengths for the series of short poly-L-proline peptides were found to be size-dependent with values of ~190 Å, ~67 Å, ~51 Å, and ~76 Å for n = 8, 12, 15, and 24, respectively. A comprehensive computational modeling was carried out to gain further insights for this surprising discovery. It was found that P8 exists as the extended all-trans isomaer whereas P12 and P15 predominantly contained one proline residue in the cis conformation. P24 exists as a mixture of one-cis (75%) and two-cis (25%) isomers where each isomer contributes to an experimentally resolvable conformational mode. This work demonstrates the resolving power of the distribution-based approach, and the capacity of integrating high-resolution single-molecule FRET experiments with molecular modeling to reveal detailed structural information about the conformation of molecules on the length scales relevant to the study of biological molecules.

Different states of synaptotagmin regulate evoked versus spontaneous release

“…To address this, we first replaced the native linker that connects these domains (residues 264–272, SAEKEEQEK) with a nine-residue poly-proline segment (9Pro). As outlined above, poly-proline was used because the side chain of this residue merges into its backbone, so the dihedral angles are relatively stable, with Φ=−78° and Ψ=+146°, resulting in a rigid helix with a periodicity of three 33 34 35 ( Fig. 1a ).…”

“…Proline is a unique amino acid; its side-chain merges with its backbone, thus restricting the backbone dihedral angles to a limited range 33 34 . These properties underlie the finding that short poly-proline segments form rigid helices with a periodicity of three (note: the structures of longer poly-proline motifs, beyond 12–15 residues, can be complex because of the increasing probability of introducing cis proline conformations 35 ). Because of this rigidity, short poly-proline rods have been used as spectroscopic rulers, or rigid spacers, for decades 34 35 36 37 .…”

“…These properties underlie the finding that short poly-proline segments form rigid helices with a periodicity of three (note: the structures of longer poly-proline motifs, beyond 12–15 residues, can be complex because of the increasing probability of introducing cis proline conformations 35 ). Because of this rigidity, short poly-proline rods have been used as spectroscopic rulers, or rigid spacers, for decades 34 35 36 37 . However, the periodicity of these helices has not been extensively exploited to address protein function and engineering problems.…”

Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker

“…Recent studies demonstrated that the end-to-end distances of the poly-L-proline peptides are shorter than those of perfectly rigid type II proline helices, probably because of the small number of cis isomers interspersed in aqueous solutions of poly-L-proline peptides [36–39]. Nevertheless, the end-to-end distance was reported to increase with an increase in the number of proline residues, up to at least 24 amino acids [38]. Therefore, the repeat number of the Pro residue in the linker is expected to control the distance between PCNA2 and PdX, and to subsequently affect the spatial arrangement of PdX relative to P450cam in PUPPET-P n .…”

“…We also estimated that the mean end-to-end distance of helical linker Pro 20 was approximately 50 Å according to a previous study [38]. PdX and P450cam are located on the same side of the PCNA ring because they are fused to the C-termini of PCNA2 and PCNA3, respectively.…”