Quantifying residue-specific conformational dynamics of a highly reactive 29-mer peptide

Abstract

Understanding structural transitions within macromolecules remains an important challenge in biochemistry, with important implications for drug development and medicine. insight into molecular behavior often requires residue-specific dynamics measurement at micromolar concentrations. We studied MP01-Gen4, a library peptide selected to rapidly undergo bioconjugation, by using electron paramagnetic resonance (epR) to measure conformational dynamics. We mapped the dynamics of MP01-Gen4 with residue-specificity and identified the regions involved in a structural transformation related to the conjugation reaction. Upon reaction, the conformational dynamics of residues near the termini slow significantly more than central residues, indicating that the reaction induces a structural transition far from the reaction site. Arrhenius analysis demonstrates a nearly threefold decrease in the activation energy of conformational diffusion upon reaction (8.0 k B T to 3.4 k B T), which occurs across the entire peptide, independently of residue position. this novel approach to epR spectral analysis provides insight into the positional extent of disorder and the nature of the energy landscape of a highly reactive, intrinsically disordered library peptide before and after conjugation.

Evolution of CPEB4 Dynamics Across its Liquid–Liquid Phase Separation Transition

“…LLPS of CPEB4 NTD exhibits a LCST behavior, where droplets exist above T t ( 10 , 60 − 62 ) and previous studies have shown that LCST is driven by hydrophobic interactions and a dominant positive entropy term. 10 , 23 , 37 , 61 − 64 Moreover, Phe and Tyr have been predicted 65 and shown to play key role in the intermolecular interaction in LLPS, 5 , 37 , 11 , 15 , 61 , 66 , 67 which includes transient interactions via intermolecular charge−π and π–π interactions. We hypothesize that these hydrophobic and aromatic interactions can lead to entropy driven LLPS of CPEB4 NTD .…”

Dynamics in supramolecular nanomaterials

“…Electron paramagnetic resonance (EPR), enabled by site-directed spin labeling (SDSL), has received attention as a suitable technique that overcomes these limitations 61 . EPR spectroscopy is highly sensitive, requiring only small concentrations of spin label, and provides a precise measure of local dynamics via the rotational diffusion rate (typically on the order of 10 6 -10 9 Hz) of the radical nitroxyl spin label [62][63] .While the synthesis of stable spin-labeled molecules and technical data analysis previously precluded the implementation of EPR, recent developments in synthesis [64][65][66] and software [67][68] have made EPR accessible for investigating conformational dynamics. EPR has been used for measuring conformational dynamics on a self-assembled nanofibers composed of peptide amphiphiles (PAs).…”

A Global Minimization Toolkit for Batch-Fitting and χ2 Cluster Analysis of CW-EPR Spectra

“…The dynamic behavior of biomolecules and soft materials is often critical to their function (1)(2)(3)(4)(5). In protein biology, for instance, a dynamic picture of proteins has gradually replaced the purely structural ''lock-and-key'' model describing their function.…”

“…Despite the availability of fitting programs, challenges remain in the analysis of dynamics by CW-EPR. Expert analysis is typically necessary to reliably estimate fit uncertainty, as well to obtain self-consistent results in variable temperature experiments (4). Such analysis reduces the risk of overfitting and improves the reproducibility and consistency of fits.…”