Unfolded Helix Research Articles

An Atomic and Molecular Insight into How PFOA Reduces α-Helicity, Compromises Substrate Binding, and Creates Binding Pockets in a Model Globular Protein.

Per- and polyfluoroalkyl substances (PFAS) pose significant health risks due to their widespread presence in various environmental and biological matrices. However, the molecular-level mechanisms underlying the interactions between PFAS and biological constituents, including proteins, carbohydrates, lipids, and DNA, remain poorly understood. Here, we investigate the interactions between a legacy PFAS, viz. perfluorooctanoic acid (PFOA), and the milk protein β-lactoglobulin (BLG) obtained using a combination of experimental and computational techniques. Circular dichroism studies reveal that PFOA perturbs the secondary structure of BLG, by driving a dose-dependent loss of α-helicity and alterations in its β-sheet content. Furthermore, exposure of the protein to PFOA attenuates the on-rate constant for the binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid (ANS), suggesting potential functional impairment of BLG by PFOA. Steered molecular dynamics and umbrella sampling calculations reveal that PFOA binding leads to the formation of an energetically favorable novel binding pocket within the protein, when residues 129-142 are steered to unfold from their initial α-helical structure, wherein a host of intermolecular interactions between PFOA and BLG's residues serve to insert the PFOA into the region between the unfolded helix and beta-sheets. Together, the data provide a novel understanding of the atomic and molecular mechanism(s) by which PFAS modulates structure and function in a globular protein, leading to a beginning of our understanding of altered biological outcomes.

A Pinch Test to Correct an Unfolded Helix: An Innovative and Predictable Technique.

The unfolded helix is an ear deformity more frequent than expected. It might be presented alone or combined with other scapha or concha anomalies, giving the appearance of big and prominent ears. Currently, there are few reports published about its restoration and few modifications have been made to its original surgical approaches since the 1970s. The technique herein described aims to facilitate the helix rim restoration procedure by considering the following preoperative and surgical advices. The preoperative use of the pinch test allows to better understand the patients' expectations, helps to explain the future outcomes to them and simplifies the marking of the tissue to be removed. The additional bilateral only-cartilage resections and the maintenance of the skin excess permit to optimize the cosmetic outcomes. This innovative surgical approach for the helix rim restoration is reliable, has little morbidity associated and presents consistent effective results. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Low-Profile Helical Quasi-Yagi Antenna Array With Multibeams at the Endfire Direction

This letter presents a novel helical quasi-Yagi antenna array with low profile and multibeams at the endfire direction. First, a planar normal-mode helical quasi-Yagi antenna with top-hat loading for low profile is designed to achieve endfire radiation. It is mounted on a conducting plane, consisting of a top-loaded folded helix as the driven element and three top-loaded unfolded helices as parasitic elements. A broadband can be achieved by controlling the resonance difference between the driven and parasitic elements. Second, a microstrip beamforming network based on Butler matrix is designed to feed a 1 × 4 helical quasi-Yagi array. The proposed antenna array is fabricated and tested. Measured results show that three endfire beams at ϕ = 90°, (90 ± 47)° are obtained while the antenna has a very low profile of 0.054 λ at its center frequency, showing that the antenna may be useful in many airborne applications such as unmanned aerial vehicles.

Revealing structural changes of prion protein during conversion from α-helical monomer to β-oligomers by means of ESR and nanochannel encapsulation.

Under nondenaturing neutral pH conditions, full-length mouse recombinant prion protein lacking the only disulfide bridge can spontaneously convert from an α-helical-dominant conformer (α-state) to a β-sheet-rich conformer (β-state), which then associates into β-oligomers, and the kinetics of this spontaneous conversion depends on the properties of the buffer used. The molecular details of this structural conversion have not been reported due to the difficulty of exploring big protein aggregates. We introduced spin probes into different structural segments (three helices and the loop between strand 1 and helix 1), and employed a combined approach of ESR spectroscopy and protein encapsulation in nanochannels to reveal local structural changes during the α-to-β transition. Nanochannels provide an environment in which prion protein molecules are isolated from each other, but the α-to-β transition can still occur. By measuring dipolar interactions between spin probes during the transition, we showed that helix 1 and helix 3 retained their helicity, while helix 2 unfolded to form an extended structure. Moreover, our pulsed ESR results allowed clear discrimination between the intra- and intermolecular distances between spin labeled residues in helix 2 in the β-oligomers, making it possible to demonstrate that the unfolded helix 2 segment lies at the association interface of the β-oligomers to form cross-β structure.

Multiscale Simulations of Diphtheria Toxin T-Domain Membrane Association

Diphtheria toxin translocation (T) domain is an alpha-helical bundle at neutral pH, which upon acidification refolds, inserts into the lipid bilayer and translocates the attached catalytic domain across the endosomal membrane. Recently, we have used a combination of computational and experimental approaches to characterize the conformation of the membrane-competent state of the T-domain, formed in solution as a result of histidine protonation [1]. Here, we model the T-domain membrane binding using a MARTINI coarse-grained (CG) model of the membrane-competent state of the protein and POPC/POPG lipid bilayers. Multi-microsecond coarse-grained molecular dynamics (MD) simulations have shown two preferred orientations of T-domain bound to the membrane interface. We will also present the results of atomic resolution refinement of these protein-lipid assemblies through approximately 10 μs-long MD simulations. An improved lipid force field model has been designed for these simulations. In both membrane bound models, T-domain slightly inserted into a POPG lipid bilayer while maintaining its orientation relative to the membrane normal. We focused our studies in a membrane bound model where partially unfolded helices TH1-4 are facing the lipid bilayer interface. Approximately 5.5 μs-long MD simulations showed little change of the T-domain orientation. However, neutralization of all acidic residues Asp and Glu followed by 3.5 μs-long MD simulations showed T-domain reorientation with helices TH8-9 aligned to the membrane plane and further protein insertion into the lipid bilayer. This suggests that protonation of acidic residues on the membrane interface may facilitate protein conformational changes leading to the final transmembrane state of helices TH8-9.Supported by NIH grant GM069783, and National Resource for Biomedical Supercomputing through NIH grant P41GM103712-S1. The Anton machine was generously made available by D.E. Shaw Research.[1] Kurnikov et al. (2013) JMB. 425:2752-2764.

Molecular dynamics simulations show how the FMRP Ile304Asn mutation destabilizes the KH2 domain structure and affects its function

Mutations or deletions of FMRP, involved in the regulation of mRNA metabolism in brain, lead to the Fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. A severe manifestation of the disease has been associated with the Ile304Asn mutation, located on the KH2 domain of the protein. Several hypotheses have been proposed to explain the possible molecular mechanism responsible for the drastic effect of this mutation in humans. Here, we performed a molecular dynamics simulation and show that the Ile304Asn mutation destabilizes the hydrophobic core producing a partial unfolding of two α-helices and a displacement of a third one. The affected regions show increased residue flexibility and motion. Molecular docking analysis revealed strongly reduced binding to a model single-stranded nucleic acid in agreement with known data that the two partially unfolded helices form the RNA-binding surface. The third helix, which we show here to be also affected, is involved in the PAK1 protein interaction. These two functional binding sites on the KH2 domain do not overlap spatially, and therefore, they can simultaneously bind their targets. Since the Ile304Asn mutation affects both binding sites, this may justify the severe clinical manifestation observed in the patient in which both mRNA metabolism activity and cytoskeleton remodeling would be affected.

Exploration of Protein Conformational Change with PELE and Meta-Dynamics.

Atomistic molecular simulation methods are now able to explore complex protein or protein-ligand dynamical space in a tractable way with methods such as meta-dynamics or adaptive biasing force. However, many of these methods either require a careful selection of reaction coordinates or the knowledge of an initial pathway of some kind. Thus, it is important that effective methods are developed to produce this pathway data in an efficient fashion. PELE, a proven protein-ligand sampling code, has been developed to provide rapid protein sampling in highly flexible cases, using a reduced network model eigen problem approach. The resulting method is able to rapidly sample configuration space with very general driving information. When applied to ubiquitin, PELE was able to reproduce RMSD and average force data found in molecular dynamics simulations. PELE was also applied to explore the opening/closing transition of T4 lysozyme. A meta-dynamics exploration using a low energy pathway validated that the configurations explored by PELE represent the most populated regions of phase space. PELE and meta-dynamics explorations also discovered a low free energy region where a large cross-domain helix of T4 lysozyme is broken in two. There is previous NMR evidence for the validity of this unfolded helix region.

Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif: A STOPPED-FLOW CIRCULAR DICHROISM KINETIC ANALYSIS

Two c-Jun leucine zipper variants that bind with high affinity to c-Fos have been selected using semirational design combined with protein-fragment complementation assays (JunW) or phage display selection (JunW(Ph1)). Enriched winners differ from each other in only two of ten semi-randomized positions, with DeltaT(m) values of 28 degrees C and 37 degrees C over wild-type. cFos-JunW, cFos-JunW(Ph1), and two intermediate mutants (cFos-JunW(Q21R) and cFos-JunW(E23K)) display biphasic kinetics in the folding direction, indicating the existence of a folding intermediate. The first reaction phase is fast and concentration-dependent, showing that the intermediate is readily populated and dimeric. The second phase is independent of concentration and is exponential. In contrast, in the unfolding direction, all molecules display two-state kinetics. Collectively this implies a transition state between unfolded helices and dimeric intermediate that is readily traversed in both directions. We demonstrate that the added stability of cFos-JunW(Ph1) relative to cFos-JunW is achieved via a combination of kinetic rate changes; cFos-JunW(E23K) has an increased initial dimerization rate, prior to the major transition state barrier while cFos-JunW(Q21R) displays a decreased unfolding rate. The former implies that improved hydrophobic burial and helix-stabilizing mutations exert their effect on the initial, rapid, monomer-collision event. In contrast, electrostatic interactions exert their effect late in the folding pathway. Although our focus is the leucine zipper region of the oncogenic transcription factor Activator Protein-1, coiled coils are ubiquitous and highly specific in their recognition of partners. Consequently, generating kinetic-based rules to predict and engineer their stability is of major significance in peptide-based drug design and nano-biotechnology.

Unfolding a Linker between Helical Repeats

In many multi-repeat proteins, linkers between repeats have little secondary structure and place few constraints on folding or unfolding. However, the large family of spectrin-like proteins, including alpha-actinin, spectrin, and dystrophin, share three-helix bundle, spectrin repeats that appear in crystal structures to be linked by long helices. All of these proteins are regularly subjected to mechanical stress. Recent single molecule atomic force microscopy (AFM) experiments demonstrate not only forced unfolding but also simultaneous unfolding of tandem repeats at finite frequency, which suggests that the contiguous helix between spectrin repeats can propagate a cooperative helix-to-coil transition. Here, we address what happens atomistically to the linker under stress by steered molecular dynamics simulations of tandem spectrin repeats in explicit water. The results for alpha-actinin repeats reveal rate-dependent pathways, with one pathway showing that the linker between repeats unfolds, which may explain the single-repeat unfolding pathway observed in AFM experiments. A second pathway preserves the structural integrity of the linker, which explains the tandem-repeat unfolding event. Unfolding of the linker begins with a splay distortion of proximal loops away from hydrophobic contacts with the linker. This is followed by linker destabilization and unwinding with increased hydration of the backbone. The end result is an unfolded helix that mechanically decouples tandem repeats. Molecularly detailed insights obtained here aid in understanding the mechanical coupling of domain stability in spectrin family proteins.

A conserved helix-unfolding motif in the naturally unfolded proteins.

Among the naturally unfolded proteins there are many polypeptides that retain an extended conformation in the absence of any apparent signal. Using sequence alignment and secondary structure prediction tools, a conserved (LS/SL)(D/E)(D/E)(D/E)X(E/D) motif is uncovered in the vicinity of the N-terminus of their unfolded helices. A comparison of these data with published observations allows one to propose that the (LS/SL)(D/E)(D/E)(D/E)X(E/D) motif is a helix-unfolding signal. Furthermore, the strong similarity between this motif and the STXXDE casein kinase II phosphorylation site suggests a regulatory mechanism for the naturally unfolded proteins within the cell.

Modeling microdomains: the surface area of globin helices.

The accessible surface areas of 53 high-resolution globin helices are correlated with molecular weight. The linear fit is assessed for statistical accuracy using a boot-strap analysis, and by comparison to the areas of 13 ideal polyalanine alpha-helices. The accessible area of the unfolded helices is compared with the folded values before helix-helix packing. An analytical physical model is presented to explain the correlation, and to provide an analytical value for the surface area parameter in the diffusion-collision model of protein folding.