Heat treatment of thioredoxin fusions increases the purity of α-helical transmembrane protein constructs.

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α-helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute-timescale heat treatment of Trx-tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single-step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches

Complex Unfolding Kinetics of Single-Domain Proteins in the Presence of Force

Single-molecule force spectroscopy is providing unique, and sometimes unexpected, insights into the free-energy landscapes of proteins. Despite the complexity of the free-energy landscapes revealed by mechanical probes, forced unfolding experiments are often analyzed using one-dimensional models that predict a logarithmic dependence of the unfolding force on the pulling velocity. We previously found that the unfolding force of the protein filamin at low pulling speed did not decrease logarithmically with the pulling speed. Here we present results from a large number of unfolding simulations of a coarse-grain model of the protein filamin under a broad range of constant forces. These show that a two-path model is physically plausible and produces a deviation from the behavior predicted by one-dimensional models analogous to that observed experimentally. We also show that the analysis of the distributions of unfolding forces (p[F]) contains crucial and exploitable information, and that a proper description of the unfolding of single-domain proteins needs to account for the intrinsic multidimensionality of the underlying free-energy landscape, especially when the applied perturbation is small

Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology

Single-molecule approaches reveal outer membrane protein biogenesis dynamics

Outer membrane proteins (OMPs) maintain the viability of Gram-negative bacteria by functioning as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of OMPs involves synchronized action of chaperones and multi-protein machineries which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Previous studies have identified proteins and their involvement along the OMP biogenesis pathway. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical cellular energy source of ATP, but solely based on thermodynamic principles, are still not well understood. Here, we highlight how different single-molecule studies can shed additional light on the mechanisms and kinetics of OMP biogenesis

Efficient Formation of Site-Specific Protein–DNA Hybrids Using Copper-Free Click Chemistry

Protein–DNA hybrids have become increasingly popular molecular building blocks in bionanotechnology and single-molecule studies to synergistically combine the programmability of DNA with the chemical diversity of proteins. The growing demand for protein–DNA hybrids requires powerful strategies for their conjugation. Here, we present an efficient two-step method for protein–DNA assembly based on copper-free click chemistry. The method allows site-specificity and high coupling efficiency, while maintaining the conservation of protein activity. We compare our method to a commonly used protocol of direct linkage of maleimide-modified oligos. We demonstrate the significantly higher yield with a protein–DNA conjugate, which is analyzed using single-molecule force spectroscopy

Structural and Optical Properties of p-InP(100) Anodized in Halogenic Acids

NRC publication: Ye