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

Unilateral heat accelerates bone elongation and lengthens extremities of growing mice

Linear growth failure results from a broad spectrum of systemic and local disorders that can generate chronic musculoskeletal disability. Current bone lengthening protocols involve invasive surgeries or drug regimens, which are only partially effective. Exposure to warm ambient temperature during growth increases limb length, suggesting that targeted heat could noninvasively enhance bone elongation. We tested the hypothesis that daily heat exposure on one side of the body unilaterally increases femoral and tibial lengths. Mice (N = 20) were treated with 40 °C unilateral heat for 40 min/day for 14 days post-weaning. Non-treated mice (N = 6) served as controls. Unilateral increases in ear (8.8%), hindfoot (3.5%), femoral (1.3%), and tibial (1.5%) lengths were obtained. Tibial elongation rate was \u3e 12% greater (15 μm/day) on the heat-treated side. Extremity lengthening correlated with temperature during treatment. Body mass and humeral length were unaffected. To test whether differences persisted in adults, mice were examined 7-weeks post-treatment. Ear area, hindfoot, femoral, and tibial lengths were still significantly increased ∼6%, 3.5%, 1%, and 1%, respectively, on the heat-treated side. Left-right differences were absent in non-treated controls, ruling out inherent side asymmetry. This model is important for designing noninvasive heat-based therapies to potentially combat a range of debilitating growth impediments in children

Transcription factor Sox10 orchestrates activity of a neural crest-specific enhancer in the vicinity of its gene

The Sox10 transcription factor is a central regulator of vertebrate neural crest and nervous system development. Its expression is likely controlled by multiple enhancer elements, among them U3 (alternatively known as MCS4). Here we analyze U3 activity to obtain deeper insights into Sox10 function and expression in the neural crest and its derivatives. U3 activity strongly depends on the presence of Sox10 that regulates its own expression as commonly observed for important developmental regulators. Sox10 bound directly as monomer to at least three sites in U3, whereas a fourth site preferred dimers. Deletion of these sites efficiently reduced U3 activity in transfected cells and transgenic mice. In stimulating the U3 enhancer, Sox10 synergized with many other transcription factors present in neural crest and developing peripheral nervous system including Pax3, FoxD3, AP2α, Krox20 and Sox2. In case of FoxD3, synergism involved Sox10-dependent recruitment to the U3 enhancer, while Sox10 and AP2α each had to bind to the regulatory region. Our study points to the importance of autoregulatory activity and synergistic interactions for maintenance of Sox10 expression and functional activity of Sox10 in the neural crest regulatory network

Theory of mechanical unfolding of homopolymer globule: all-or-none transition in force-clamp mode vs phase coexistence in position-clamp mode

Equilibrium mechanical unfolding of a globule formed by long flexible homopolymer chain collapsed in a poor solvent and subjected to an extensional force f (force-clamp mode) or extensional deformation D (position-clamp mode) is studied theoretically. Our analysis, like all previous analysis of this problem, shows that the globule behaves essentially differently in two modes of extension. In the force-clamp mode, mechanical unfolding of the globule with increasing applied force occurs without intramolecular microphase segregation, and at certain threshold value of the pulling force the globule unfolds as a whole ("all-or-none" transition). The value of the threshold force and the corresponding jump in the distance between the chain ends increase with a deterioration of the solvent quality and/or with an increase in the degree of polymerization. In the position-clamp mode, the globule unfolding occurs via intramolecular microphase coexistence of globular and extended microphases followed by an abrupt unraveling transition. Reaction force in the microphase segregation regime demonstrates an "anomalous" decrease with increasing extension. Comparison of deformation curves in force and position-clamp modes demonstrates that at weak and strong extensions the curves for two modes coincide, differences are observed in the intermediate extension range. Another unfolding scenario is typical for short globules: in both modes of extension they unfold continuously, without jumps or intramolecular microphase coexistence, by passing a sequence of uniformly elongated configurations.Comment: 19 pages, 13 figures, 1 tabl

Influence of Hydrodynamic Interactions on Mechanical Unfolding of Proteins

We incorporate hydrodynamic interactions in a structure-based model of ubiquitin and demonstrate that the hydrodynamic coupling may reduce the peak force when stretching the protein at constant speed, especially at larger speeds. Hydrodynamic interactions are also shown to facilitate unfolding at constant force and inhibit stretching by fluid flows.Comment: to be published in Journal of Physics: Condensed Matte

Knotting and unknotting of a protein in single molecule experiments.

Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.N.C.H.L. is supported by a UBD Chancellor’s Scholarship from the Brunei Government. W.N. was supported by the Ministry of Education (MoE) Singapore. S.S.M acknowledges funding from the Alexander von Humboldt Foundation. This work was supported by an SFB 863 A2 grant of Deutsche Forschungsgemeinschaft to M.R

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

The transcription factor Sox5 modulates Sox10 function during melanocyte development

The transcription factor Sox5 has previously been shown in chicken to be expressed in early neural crest cells and neural crest-derived peripheral glia. Here, we show in mouse that Sox5 expression also continues after neural crest specification in the melanocyte lineage. Despite its continued expression, Sox5 has little impact on melanocyte development on its own as generation of melanoblasts and melanocytes is unaltered in Sox5-deficient mice. Loss of Sox5, however, partially rescued the strongly reduced melanoblast generation and marker gene expression in Sox10 heterozygous mice arguing that Sox5 functions in the melanocyte lineage by modulating Sox10 activity. This modulatory activity involved Sox5 binding and recruitment of CtBP2 and HDAC1 to the regulatory regions of melanocytic Sox10 target genes and direct inhibition of Sox10-dependent promoter activation. Both binding site competition and recruitment of corepressors thus help Sox5 to modulate the activity of Sox10 in the melanocyte lineage

Role of conservative mutations in protein multi-property adaptation

Protein physicochemical properties must undergo complex changes during evolution, as a response to modifications in the organism environment, the result of the proteins taking up new roles or because of the need to cope with the evolution of molecular interacting partners. Recent work has emphasized the role of stability and stability–function trade-offs in these protein adaptation processes. In the present study, on the other hand, we report that combinations of a few conservative, high-frequency-of-fixation mutations in the thioredoxin molecule lead to largely independent changes in both stability and the diversity of catalytic mechanisms, as revealed by single-molecule atomic force spectroscopy. Furthermore, the changes found are evolutionarily significant, as they combine typically hyperthermophilic stability enhancements with modulations in function that span the ranges defined by the quite different catalytic patterns of thioredoxins from bacterial and eukaryotic origin. These results suggest that evolutionary protein adaptation may use, in some cases at least, the potential of conservative mutations to originate a multiplicity of evolutionarily allowed mutational paths leading to a variety of protein modulation patterns. In addition the results support the feasibility of using evolutionary information to achieve protein multi-feature optimization, an important biotechnological goal