Backbone Folding Research Articles

Azobenzene-Based Main-Chain Liquid Crystalline Polymers Bearing Periodically Installed Alkyl, PEG, or Fluoroalkyl Segments and Photoisomerization Studies

ABSTRACT In the present study, liquid crystalline polymers (LCPs) were prepared by melt-transesterification of azobenzene-bearing diols and diethyl malonate (DEM) derivatized monomers. The effect of the nature and length of the installed pendant segments on the mesomorphic properties of the prepared LCPs was investigated by differential scanning calorimetry (DSC), X-ray diffraction (XRD), and polarized optical microscopy (POM) studies. The formation of layered morphology was inferred from X-ray scattering analysis arising from the colocation of rigid azobenzene moieties in one layer and the flexible pendant segments in alternate layers due to the zigzag folding of the polymer backbone. The linear variation of the interlamellar spacing with the number of carbon atoms in the pendant alkyl segments corroborates the proposed zigzag backbone folding in the polymers. The rate constant values obtained from the photoisomerization studies of the isomeric polymers and model compound, which are in the same ballpark, reveal that the photoisomerization process of such polymers is not affected by the position or size of the azobenzene unit.

Heat-activated growth of metastable and length-defined DNA fibers expands traditional polymer assembly

Biopolymers such as nucleic acids and proteins exhibit dynamic backbone folding, wherein site-specific intramolecular interactions determine overall structure. Proteins then hierarchically assemble into supramolecular polymers such as microtubules, that are robust yet dynamic, constantly growing or shortening to adjust to cellular needs. The combination of dynamic, energy-driven folding and growth with structural stiffness and length control is difficult to achieve in synthetic polymer self-assembly. Here we show that highly charged, monodisperse DNA-oligomers assemble via seeded growth into length-controlled supramolecular fibers during heating; when the temperature is lowered, these metastable fibers slowly disassemble. Furthermore, the specific molecular structures of oligomers that promote fiber formation contradict the typical theory of block copolymer self-assembly. Efficient curling and packing of the oligomers – or ‘curlamers’ – determine morphology, rather than hydrophobic to hydrophilic ratio. Addition of a small molecule stabilises the DNA fibers, enabling temporal control of polymer lifetime and underscoring their potential use in nucleic-acid delivery, stimuli-responsive biomaterials, and soft robotics.

Effects of altered backbone composition on the folding kinetics and mechanism of an ultrafast-folding protein.

Sequence-encoded protein folding is a ubiquitous biological process that has been successfully engineered in a range of oligomeric molecules with artificial backbone chemical connectivity. A remarkable aspect of protein folding is the contrast between the rapid rates at which most sequences in nature fold and the vast number of conformational states possible in an unfolded chain with hundreds of rotatable bonds. Research efforts spanning several decades have sought to elucidate the fundamental chemical principles that dictate the speed and mechanism of natural protein folding. In contrast, little is known about how protein mimetic entities transition between an unfolded and folded state. Here, we report effects of altered backbone connectivity on the folding kinetics and mechanism of the B domain of Staphylococcal protein A (BdpA), an ultrafast-folding sequence. A combination of experimental biophysical analysis and atomistic molecular dynamics simulations performed on the prototype protein and several heterogeneous-backbone variants reveal the interplay among backbone flexibility, folding rates, and structural details of the transition state ensemble. Collectively, these findings suggest a significant degree of plasticity in the mechanisms that can give rise to ultrafast folding in the BdpA sequence and provide atomic level insights into how protein mimetic chains adopt an ordered folded state.

Hard-Soft Cooperativity of an Allosteric Tweezer.

Drawing inspiration from allosteric proteins, a zigzag-shaped π-conjugation was structurally engineered into a tweezer-like ionophore having multiple disparate binding sites. When a soft metal ion binds to the central tridentate ligand motif, the rigid backbone folds, bringing two macrocyclic arms into close proximity. Stabilized by a coordinating anion, the tweezer-like conformation of the resulting metalloligand recruits a hard cation to form a sandwich-like complex with a remarkably enhanced binding affinity and selectivity.

OPUS-Fold3: a gradient-based protein all-atom folding and docking framework on TensorFlow.

For refining and designing protein structures, it is essential to have an efficient protein folding and docking framework that generates a protein 3D structure based on given constraints. In this study, we introduce OPUS-Fold3 as a gradient-based, all-atom protein folding and docking framework, which accurately generates 3D protein structures in compliance with specified constraints, such as a potential function as long as it can be expressed as a function of positions of heavy atoms. Our tests show that, for example, OPUS-Fold3 achieves performance comparable to pyRosetta in backbone folding and significantly better in side-chain modeling. Developed using Python and TensorFlow 2.4, OPUS-Fold3 is user-friendly for any source-code level modifications and can be seamlessly combined with other deep learning models, thus facilitating collaboration between the biology and AI communities. The source code of OPUS-Fold3 can be downloaded from http://github.com/OPUS-MaLab/opus_fold3. It is freely available for academic usage.

Computational Study of Helical and Helix-Hinge-Helix Conformations of an Anti-Microbial Peptide in Solution by Molecular Dynamics and Vibrational Analysis.

Many classical antimicrobial peptides adopt an amphipathic helical structure at a water-membrane interface. Prior studies led to the hypothesis that a hinge near the middle of a helical peptide plays an important role in facilitating peptide-membrane interactions. Here, dynamics and vibrations of a designed hybrid antimicrobial peptide LM7-2 in solution were simulated to investigate its hinge formation. Molecular dynamics simulation results on the basis of the CHARMM36 force field showed that the α-helix LM7-2 bent around two or three residues near the middle of the peptide, stayed in a helix-hinge-helix conformation for a short period of time, and then returned to a helical conformation. High-resolution computational vibrational techniques were applied on the LM7-2 system when it has α-helical and helix-hinge-helix conformations to understand how this structural change affects its inherent vibrations. These studies concentrated on the calculation of frequencies that correspond to backbone amide bands I, II, and III: vibrational modes that are sensitive to changes in the secondary structure of peptides and proteins. To that end, Fourier transforms were applied to thermal fluctuations in C-N-H angles, C-N bond lengths, and C═O bond lengths of each amide group. In addition, instantaneous all-atom normal mode analysis was applied to monitor and detect the characteristic amide bands of each amide group within LM7-2 during the MD simulation. Computational vibrational results indicate that shapes and frequencies of amide bands II and especially III were altered only for amide groups near the hinge. These methods provide high-resolution vibrational information that can complement spectroscopic vibrational studies. They assist in interpreting spectra of similar systems and suggest a marker for the presence of the helix-hinge-helix motif. Moreover, radial distribution functions indicated an increase in the probability of hydrogen bonding between water and a hydrogen atom connected to nitrogen (HN) in such a hinge. The probability of intramolecular hydrogen bond formation between HN and an amide group oxygen atom within LM7-2 was lower around the hinge. No correlation has been found between the presence of a hinge and hydrogen bonds between amide group oxygen atoms and the hydrogen atoms of water molecules. This result suggests a mechanism for hinge formation wherein hydrogen bonds to oxygen atoms of water replace intramolecular hydrogen bonds as the peptide backbone folds.

Unraveling Allostery in a Knotted Minimal Methyltransferase by NMR Spectroscopy

The methyltransferases that belong to the SpoU-TrmD family contain trefoil knots in their backbone fold. Recent structural dynamic and binding analyses of both free and bound homologs indicate that the knot within the polypeptide backbone plays a significant role in the biological activity of the molecule. The knot loops form the S-adenosyl-methionine (SAM)-binding pocket as well as participate in SAM binding and catalysis. Knots contain both at once a stable core as well as moving parts that modulate long-range motions. Here, we sought to understand allosteric effects modulated by the knotted topology. Uncovering the residues that contribute to these changes and the functional aspects of these protein motions are essential to understanding the interplay between the knot, activation of the methyltransferase, and the implications in RNA interactions. The question we sought to address is as follows: How does the knot, which constricts the backbone as well as forms the SAM-binding pocket with its three distinctive loops, affect the binding mechanism? Using a minimally tied trefoil protein as the framework for understanding the structure–function roles, we offer an unprecedented view of the conformational mechanics of the knot and its relationship to the activation of the ligand molecule. Focusing on the biophysical characterization of the knot region by NMR spectroscopy, we identify the SAM-binding region and observe changes in the dynamics of the loops that form the knot. Importantly, we also observe long-range allosteric changes in flanking helices consistent with winding/unwinding in helical propensity as the knot tightens to secure the SAM cofactor.

Design of a thrombin resistant human acidic fibroblast growth factor (hFGF1) variant that exhibits enhanced cell proliferation activity

Acidic fibroblast growth factors (FGF1s) are heparin binding proteins that regulate a wide array of key cellular processes and are also candidates for promising biomedical applications. FGF1-based therapeutic applications are currently limited due to their inherent thermal instability and susceptibility to proteases. Using a wide range of biophysical and biochemical techniques, we demonstrate that reversal of charge on a well-conserved positively charged amino acid, R136, in the heparin binding pocket drastically increases the resistance to proteases, thermal stability, and cell proliferation activity of the human acidic fibroblast growth factor (hFGF1). Two-dimensional NMR data suggest that the single point mutations at position-136 (R136G, R136L, R136Q, R136K, and R136E) did not perturb the backbone folding of hFGF1. Results of the differential scanning calorimetry experiments show that of all the designed R136 mutations only the charge reversal mutation, R136E, significantly increases (ΔTm = 7 °C) the thermal stability of the protein. Limited trypsin and thrombin digestion results reveal that the R136E mutation drastically increases the resistance of hFGF1 to the action of the serine proteases. Isothermal titration calorimetry data show that the R136E mutation markedly decreases the heparin binding affinity of hFGF1. Interestingly, despite lower heparin binding affinity, the cell proliferation activity of the R136E variant is more than double of that exhibited by either the wild type or the other R136 variants. The R136E variant due to its increased thermal stability, resistance to proteases, and enhanced cell proliferation activity are expected to provide valuable clues for the development of hFGF1- based therapeutics for the management of chronic diabetic wounds.

Folding free energy landscapes of β-sheets with non-polarizable and polarizable CHARMM force fields.

Molecular dynamics (MD) simulations of peptides and proteins offer atomic-level detail into many biological processes, although the degree of insight depends on the accuracy of the force fields used to represent them. Protein folding is a key example in which the accurate reproduction of folded-state conformations of proteins and kinetics of the folding processes in simulation is a longstanding goal. Although there have been a number of recent successes, challenges remain in capturing the full complexity of folding for even secondary-structure elements. In the present work, we have used all-atom MD simulations to study the folding properties of one such element, the C-terminal β-hairpin of the B1 domain of streptococcal protein G (GB1). Using replica-exchange umbrella sampling simulations, we examined the folding free energy of two fixed-charge CHARMM force fields, CHARMM36 and CHARMM22*, as well as a polarizable force field, the CHARMM Drude-2013 model, which has previously been shown to improve the folding properties of α-helical peptides. The CHARMM22* and Drude-2013 models are in rough agreement with experimental studies of GB1 folding, while CHARMM36 overstabilizes the β-hairpin. Additional free-energy calculations show that small adjustments to the atomic polarizabilities in the Drude-2013 model can improve both the backbone solubility and folding properties of GB1 without significantly affecting the model's ability to properly fold α-helices. We also identify a non-native salt bridge in the β-turn region that overstabilizes the β-hairpin in the C36 model. Finally, we demonstrate that tryptophan fluorescence is insufficient for capturing the full β-hairpin folding pathway.

The JAVA Based Computational Tool for Pairwise Comparison of Protein Backbone Folds in Liganded and Apo 3D Structures of the alpha/beta Hydrolase Fold Proteins

Cholinesterases, carboxylesterases and lipases are some of prominent catalytically active proteins with specifically similar, globular tertiary structure fold termed “alpha/beta hydrolase fold”. Ligand binding, both reversible and covalent, frequently results in small, yet systematic and potentially functionally important changes in the conformation of their alpha carbon backbones that can be easily overseen by commonly used overlays based on overall minimization of backbone atom RMSD analysis. In particular, backbone conformations of cholinesterases ‐ acetylcholinesterase and butyrylcholinesterase ‐ show only small conformational changes. This is revealed in more than 200 PDB deposited cholinesterase X‐ray structures in complexes with structurally diverse ligands that affect their function.We developed a novel, reference point based principle for overlay‐independent pairwise comparison of liganded and non‐liganded alpha carbon backbone conformations from respective PDB (Protein Data Bank) deposited 3D structure entries and encoded it in JAVA based computer algorithm for quick analysis. Comparisons are based on differences in distances between each alpha carbon and chosen reference point in each pair of compared structures, as well as on differences in the angle between center of mass, reference point and each of alpha carbons in the comparison. Combination of the two comparison criteria reveals subset of backbone alpha carbons in two structures that maintains best their relative positions in the 3D space and that can be used as tethering points for overlay of compared structures. Created overlay has capacity to reveal small but systematic local changes in the backbone conformation frequently masked by the overall RMSD minimization approach.Our systematic analysis of a number of PDB deposited alpha/beta hydrolase fold structures revealed small, yet functionally important conformational changes in their backbone consistent with experimental observations and instrumental in revealing molecular mechanisms in respective catalytic reactions. These findings are important for complete molecular target template characterization in the structure based development of novel antidotes of exposure to organophosphates which covalently inhibit this family of enzymes.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant R21 NS098998 from NINDS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The crystal structure of the lipoaminopeptaibol helioferin, an antibiotic peptide from Mycogone rosea.

The crystal structure of the natural nonapeptide antibiotic helioferin has been determined and refined to 0.9 Å resolution. Helioferin consists of helioferin A and B, which contain 2-(2'-aminopropyl)aminoethanol (Apae) and 2-[(2'-aminopropyl)methylamino]ethanol (Amae) at their respective alkanolamine termini. In addition, helioferin contains the unusual amino-acid residues α-aminoisobutyric acid (Aib) and (2S,4S,6S)-2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (Ahmod). The amino-terminus is capped with 2-methyl-n-1-octanoic acid (M8a). The peptide crystallizes with a 1:1 molar ratio of helioferin A and B in the monoclinic space group C2, with unit-cell parameters a = 34.711, b = 10.886, c = 17.150 Å, β = 93.05°. The peptide backbone folds in a regular right-handed α-helical conformation, with eight intramolecular hydrogen bonds, all but one forming 5→1 interactions. The two aliphatic chains of the fatty-acyl (M8a) and the second residue (Ahmod) extend out of the α-helical structure in opposite directions and lead to a corkscrew-like shape of the peptide molecule. Halogen anions (Cl- and F-) have been co-crystallized with the peptide molecules, implying a positive charge at the aminoalcohol end of the peptide. In the tightly packed crystal the helices are linked head to tail via the anions by electrostatic, hydrogen-bond and van der Waals interactions, forming continuous helical rods. Two nonparallel rods (forming an angle of 118°) interact directly via hydrogen bonds and via the anions, forming a double layer. Successive double layers are held together only via van der Waals contacts. The helical axes of successive double layers are also related by an angle of 118°. The structure of helioferin reported here and the previously determined structure of the homologous leucinostatin A have a total straight length of about 21 Å, indicating a different membrane-modifying bioactivity from that of long-chain, amphiphilic peptaibols.

Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin.

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure-function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating C. botulinum infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure-function link, not only of a molecule but also of the pathogenesis.

Backbone assignments for the SPOUT methyltransferase MTT Tm , a knotted protein from Thermotoga maritima.

The SPOUT family of methyltransferase proteins is noted for containing a deep trefoil knot in their defining backbone fold. This unique fold is of high interest for furthering the understanding of knots in proteins. Here, we report the 1H, 13C, 15N assignments for MTT Tm , a canonical member of the SPOUT family. This protein is unique, as it is one of the smallest members of the family, making it an ideal system for probing the unique properties of the knot. Our present work represents the foundation for further studies into the topology of MTT Tm , and understanding how its structure affects both its folding and function.

Molecular modeling of Gly80 and Ser80 variants of human group IID phospholipase A2 and their receptor complexes: potential basis for weight loss in chronic obstructive pulmonary disease.

Weight loss is a well known systemic manifestation of chronic obstructive pulmonary disease (COPD). A Gly80Ser mutation on human group IID secretory phospholipase A2 (sPLA2) enhances expression of the cytokines that are responsible for weight loss. In this study, we seek to establish a structural correlation of wild type sPLA2 and the Gly80Ser mutation with function. sPLA2 with glycine and serine at the 80th positions and the M-type receptor were modelled. The enzymes were docked to the receptor and molecular dynamics was carried out to 70ns. Structural analysis revealed the enzymes to comprise three helices (H1-H3), two short helices (SH1 and SH2), and five loops including a calcium binding loop (L1-L5), and to be stabilized by seven disulfide bonds. The overall backbone folds of the two models are very similar, with main chain RMSD of less than 1Å. The active site within the substrate binding channel shows a catalytic triad of water-His67-Asp112, showing a hydrogen bonded network. Major structural differences between wild type and mutant enzymes were observed locally at the site of the mutation and in their global conformations. These differences include: (1) loop-L3 between H2 and H3, which bears residue Gly80 in the wild type, is in a closed conformation with respect to the channel opening, while in the mutant enzyme it adopts a relatively open conformation; (2) the mutant enzyme is less compact and has higher solvent accessible surface area; and (3) interfacial binding contact surface area is greater, and the quality of interactions with the receptor is better in the mutant enzyme as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that could determine the increased potency of the mutant enzyme with macrophage receptor for cytokine secreting function, resulting in exacerbation of cachexia in COPD.

Biophysical characterization of the domain association between cytosolic A and B domains of the mannitol transporter enzymes II(Mtl) in the presence and absence of a connecting linker.

The mannitol transporter enzyme II(Mtl) of the bacterial phosphotransferase system is a multi-domain protein that catalyzes mannitol uptake and phosphorylation. Here we investigated the domain association between cytosolic A and B domains of enzyme II(Mtl) , which are natively connected in Escherichia coli, but separated in Thermoanaerobacter tengcongensis. NMR backbone assignment and residual dipolar couplings indicated that backbone folds were well conserved between the homologous domains. The equilibrium binding of separately expressed domains, however, exhibited ∼28-fold higher affinity compared to the natively linked ones. Phosphorylation of the active site loop significantly contributed to the binding by reducing conformational dynamics at the binding interface, and a few key mutations at the interface were critical to further stabilize the complex by hydrogen bonding and hydrophobic interactions. The affinity increase implicated that domain associations in cell could be maintained at an optimal level regardless of the linker.

Xylan decoration patterns and the plant secondary cell wall molecular architecture.

The molecular architecture of plant secondary cell walls is still not resolved. There are several proposed structures for cellulose fibrils, the main component of plant cell walls and the conformation of other molecules is even less well known. Glucuronic acid (GlcA) substitution of xylan (GUX) enzymes, in CAZy family glycosyl transferase (GT)8, decorate the xylan backbone with various specific patterns of GlcA. It was recently discovered that dicot xylan has a domain with the side chain decorations distributed on every second unit of the backbone (xylose). If the xylan backbone folds in a similar way to glucan chains in cellulose (2-fold helix), this kind of arrangement may allow the undecorated side of the xylan chain to hydrogen bond with the hydrophilic surface of cellulose microfibrils. MD simulations suggest that such interactions are energetically stable. We discuss the possible role of this xylan decoration pattern in building of the plant cell wall.

Single chain structure of a poly(N-isopropylacrylamide) surfactant in water.

We present atomistic simulations of a single PNIPAM-alkyl copolymer surfactant in aqueous solution at temperatures below and above the LCST of PNIPAM. We compare properties of the surfactant with pure PNIPAM oligomers of similar lengths, such as the radius of gyration and solvent accessible surface area, to determine the differences in their structures and transition behavior. We also explore changes in polymer-polymer and polymer-water interactions, including hydrogen bond formation. The expected behavior is observed in the pure PNIPAM oligomers, where the backbone folds onto itself above the LCST in order to shield the hydrophobic groups from water. The surfactant, on the other hand, does not show much conformational change as a function of temperature, but instead folds to bring the hydrophobic alkyl tail and PNIPAM headgroup together at all temperatures. The atomic detail available from these simulations offers important insight into understanding how the transition behavior is changed in PNIPAM-based systems.

Backbone fold of Human Small Ubiquitin like Modifier protein-1 (SUMO-1) based on Prot3D-NMR approach.

Backbone fold of Human Small Ubiquitin like Modifier protein-1 (SUMO-1) based on Prot3D-NMR approach.

What is the shape of the distribution of protein conformations at equilibrium?

According to the thermodynamic hypothesis, the native state of proteins is that in which the free energy of the system is at its lowest, so that at normal temperature and pressure, proteins evolve to that state. We selected four proteins representative of each of the four classes, and for each protein make four simulations, one starting from the native structure and the other three starting from the structure obtained by threading the sequence of one protein onto the native backbone fold of the other three proteins. Because of their large conformational distances with respect to the native structure, the three alternative initial structures cannot be considered as local minima within the native ensemble of the corresponding protein. As expected, the initial native states are preserved in the .5 μs simulations performed here and validate the simulations. On the other hand, when the initial state is not native, an analysis of the trajectories does not reveal any evolution towards the native state, during that time. These results indicate that the distribution of protein conformations is multipeak shaped, so that apart from the peak corresponding to the native state, there are other peaks associated with average structures that are very different from the native and that can last as long as the native state.

A theoretical study of the stability of disulfide bridges in various β-sheet structures of protein segment models

Electron structure calculations are used to explore stabilization effects of disulfide bridges in a (Ala–Cys–Ala–Cys–Ala)2 β-sheet model both in the parallel and the anti-parallel (103142 and 143102) arrangements. Stabilities were calculated using a redox reaction involving a weak oxidizing agent (1,4-benzoquinone). The results show that both inter- and intra-strand disulfide SS-bridges stabilize the β-sheet backbone fold. However, inter-strand SS-bridges give more stability than their intra-strand counterparts. For both single and double disulfide linked conformations, stabilization was larger for the parallel than for the anti-parallel β-sheet arrangements.