Helical Folding Research Articles

How lipids affect the energetics of co-translational alpha helical membrane protein folding.

Membrane proteins need to fold with precision in order to function correctly, with misfolding potentially leading to disease. The proteins reside within a hydrophobic lipid membrane and must insert into the membrane and fold correctly, generally whilst they are being translated by the ribosome. Favourable and unfavourable free energy contributions are present throughout each stage of insertion and folding. The unfavourable energy cost of transferring peptide bonds into the hydrophobic membrane interior is compensated for by the favourable hydrophobic effect of partitioning a hydrophobic transmembrane alpha-helix into the membrane. Native membranes are composed of many different types of lipids, but how these different lipids influence folding and the associated free energies is not well understood. Altering the lipids in the bilayer is known to affect the probability of transmembrane helix insertion into the membrane, and lipids also affect protein stability and can promote successful folding. This review will summarise the free energy contributions associated with insertion and folding of alpha helical membrane proteins, as well as how lipids can make these processes more or less favourable. We will also discuss the implications of this work for the free energy landscape during the co-translational folding of alpha helical membrane proteins.

How physical forces drive the process of helical membrane protein folding.

Protein folding is a fundamental process of life with important implications throughout biology. Indeed, tens of thousands of mutations have been associated with diseases, and most of these mutations are believed to affect protein folding rather than function. Correct folding is also a key element of design. These factors have motivated decades of research on protein folding. Unfortunately, knowledge of membrane protein folding lags that of soluble proteins. This gap is partly caused by the greater technical challenges associated with membrane protein studies, but also because of additional complexities. While soluble proteins fold in a homogenous water environment, membrane proteins fold in a setting that ranges from bulk water to highly charged to apolar. Thus, the forces that drive folding vary in different regions of the protein, and this complexity needs to be incorporated into our understanding of the folding process. Here, we review our understanding of membrane protein folding biophysics. Despite the greater challenge, better model systems and new experimental techniques are starting to unravel the forces and pathways in membrane protein folding.

Widespread genetic heterogeneity of human ribosomal RNA genes.

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5' ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5' ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.

Potassium channel folding assessed by hydrogen exchange mass spectrometry and tryptophan fluorescence identifies pore helix and selectivity filter folding events

While the function of ion channels is critical for numerous physiological processes, how they achieve their three-dimensional fold and assemble remains an open topic. Outstanding questions concern the identification of the rate-limiting step, when the selectivity filter folds, and the role of a protein-dense phase in folding. We have made significant progress in characterizing the in vitro folding of the KcsA transmembrane pore domain, a robust model system for the pore domain of human potassium channels such as hERG and Kv1.2.

Exploring Helical Folding in Oligomers of Cyclopentane-Based ϵ-Amino Acids: A Computational Study.

The conformational preferences of oligopeptides of an ϵ‐amino acid (2‐((1R,3S)‐3‐(aminomethyl)cyclopentyl)acetic acid, Amc5a) with a cyclopentane substituent in the Cβ−Cγ−Cδ sequence of the backbone were investigated using DFT methods in chloroform and water. The most preferred conformation of Amc5a oligomers (dimer to hexamer) was the H16 helical structure both in chloroform and water. Four residues were found to be sufficient to induce a substantial H16 helix population in solution. The Amc5a hexamer adopted a stable left‐handed (M)‐2.316 helical conformation with a rise of 4.8 Å per turn. The hexamer of Ampa (an analogue of Amc5a with replacing cyclopentane by pyrrolidine) adopted the right‐handed mixed (P)‐2.918/16 helical conformation in chloroform and the (M)‐2.416 helical conformation in water. Therefore, hexamers of ϵ‐amino acid residues exhibited different preferences of helical structures depending on the substituents in peptide backbone and the solvent polarity as well as the chain length.

Effect of a cis-4-aminopiperidine-3-carboxylic acid (cis-APiC) residue on mixed-helical folding of unnatural peptides.

The α/β-peptide 11/9-helix and the β-peptide 12/10-helix belong to "mixed" helices, in which two types of hydrogen bonds with opposite directionality alternate along the helical axis. cis-2-Aminocyclohexanecarboxylic acid (cis-ACHC) is known to promote these mixed helices and stabilize the helical propensity more than other acyclic β-residues. Application of a mixed-helical backbone still requires sufficient solubility in aqueous solution. In this regard, we chose cis-4-aminopiperidine-3-carboxylic acid (cis-APiC) as a foldamer building block that can provide both sufficient aqueous solubility and mixed-helical propensity. Conformational analyses of α/β- and β-peptides containing a cis-APiC residue by circular dichroism spectroscopy and single-crystal X-ray crystallography suggest that the incorporation of cis-APiC instead of cis-ACHC can enhance the aqueous solubility of the mixed-helical peptides without any adverse effect on helical folding. In addition, the ratio between right- and left-handed 12/10-helices of β-peptides can be rationalized by relative energies between the local conformations of the cis-APiC residue.

Tweezer-type binding cavity formed by the helical folding of a carbazole-pyridine oligomer.

We have synthesised a new aromatic foldamer based on the carbazole-pyridine oligomers that adopt helical conformations via dipole-dipole interactions and π-stacking between two ethynyl bond-linked monomers. This foldamer scaffold has been further modified into a synthetic receptor with a tweezer-type binding cavity outside the helical backbone upon folding, in contrast to most aromatic foldamers with internal binding cavities. The tweezer-type cavity is composed of two parallel pyrenyl planes, allowing for the intercalation of a naphthalenediimide guest via π-stacking and CH⋯O interactions, as demonstrated using its 1H NMR spectra and X-ray crystal structure.

Helical sulfono-γ-AApeptides with predictable functions in protein recognition.

Sulfono-γ-AApeptides are a subset of possible sequence-specific foldamers that might be considered for the design of biomimetic drug molecular structures. Although they have been studied for a relatively short period of time, a number of structures and functions have been designed or discovered within this class of unnatural peptides. Examples of utilizing these sulfono-γ-AApeptides have demonstrated the potential that sulfono-γ-AApeptides can offer, however, to date, their application in biomedical sciences yet remains unexplored. This review mainly summarizes the helical folding conformations of sulfono-γ-AApeptides and their biological application as helical mimetics in medicinally relevant protein–protein interactions (PPIs) and assesses their potential for the mimicry of other α-helices for protein recognition in the future.

Synthesis and conformational analysis of an anti‐β2,3‐amino acid as a building block for unnatural peptide helices

AbstractAnti‐β2,3‐amino acids are a class of acyclic β‐amino acids that usually stabilize β‐sheet‐like conformations of unnatural peptides. (2S,3R)‐3‐amino‐2‐ethylpentanoic acid (AEPA) is a diethyl‐substituted anti‐β2,3‐amino acid that can be regarded as an acyclic analog of cis‐2‐aminocyclohexanecarboxylic acid, which is known to promote the α/β‐peptide 11/9‐helix and the β‐peptide 12/10‐helix. We report that AEPA can be incorporated into the two unnatural peptide helices without disrupting helical folding. Crystal structure data reveal that the anti‐β2,3‐residue adopts unconventional gauche (+) conformation in those unnatural peptide helices.

Folding and Insertion of Transmembrane Helices at the ER.

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how transmembrane helices achieve membrane integration. We will address questions such as: Where do the stretches of amino acids fold into a helical conformation? What is/are the route/routes that these stretches take from synthesis at the ribosome to integration through the ER translocon? How do these stretches ‘know’ to integrate and in which orientation? How do marginally hydrophobic stretches of amino acids integrate and survive as transmembrane helices?

α-Helices propagating from stable nucleators exhibit unconventional thermal folding.

Although the effect of thermal perturbations on protein structure has long been modeled in helical peptides, several details, such as the relation between the thermal stabilities of the propagating and nucleating segments of helices, remain elusive. We had earlier reported on the helix-nucleating propensities of covalent H-bond surrogate-constrained α-turns. Here, we analyze the thermal stabilities of helices that propagate along peptides appended to these α-helix nucleators using their NMR and far-UV CD spectra. Unconventional thermal folding of these helix models reveals that the helical fold in propagating backbones resists thermal perturbations as long as their nucleating template is intact. The threshold temperature of such resistance is also influenced by the extent of similarity between the natures of helical folds in the nucleating and propagating segments. Correlations between helicities and rigidities of helix-nucleating and helix-propagating segments reveal subtle interdependence, which explains cooperativity and residual helix formation during protein folding.

Inside and Out of the Pore: Comparing Interactions and Molecular Dynamics of Influenza A M2 Viroporin Complexes in Standard Lipid Bilayers.

Ion channels located at viral envelopes (viroporins) have a critical function for the replication of infectious viruses and are important drug targets. Over the last decade, the number and duration of molecular dynamics (MD) simulations of the influenza A M2 ion channel owing to the increased computational efficiency. Here, we aimed to define the system setup and simulation conditions for the correct description of the protein-pore and the protein-lipid interactions for influenza A M2 in comparison with experimental data. We performed numerous MD simulations of the influenza A M2 protein in complex with adamantane blockers in standard lipid bilayers using OPLS2005 and CHARMM36 (C36) force fields. We explored the effect of varying the M2 construct (M2(22-46) and M2(22-62)), the lipid buffer size and type (stiffer DMPC or softer POPC with or without 20% cholesterol), the simulation time, the H37 protonation site (Nδ or Νε), the conformational state of the W41 channel gate, and M2's cholesterol binding sites (BSs). We report that the 200 ns MD with M2(22-62) (having Nε Η37) in the 20 Å lipid buffer with the C36 force field accurately describe: (a) the M2 pore structure and interactions inside the pore, that is, adamantane channel blocker location, water clathrate structure, and water or chloride anion blockage/passage from the M2 pore in the presence of a channel blocker and (b) interactions between M2 and the membrane environment as reflected by the calculation of the M2 bundle tilt, folding of amphipathic helices, and cholesterol BSs. Strikingly, we also observed that the C36 1 μs MD simulations using M2(22-62) embedded in a 20 Å POPC:cholesterol (5:1) scrambled membrane produced frequent interactions with cholesterol, which when combined with computational kinetic analysis, revealed the experimentally observed BSs of cholesterol and suggested three similarly long-interacting positions in the top leaflet that have previously not been observed experimentally. These findings promise to be useful for other viroporin systems.

Designing BH3-Mimetic Peptide Inhibitors for the Viral Bcl-2 Homologues A179L and BHRF1: Importance of Long-Range Electrostatic Interactions.

Viruses have evolved strategies to prevent apoptosis of infected cells at early stages of infection. The viral proteins (vBcl-2s) from specific viral genes adopt a helical fold that is structurally similar to that of mammalian antiapoptotic Bcl-2 proteins and exhibit little sequence similarity. Hence, vBcl-2 homologues are attractive targets to prevent viral infection. However, very few studies have focused on developing inhibitors for vBcl-2 homologues. In this study, we have considered two vBcl-2 homologues, A179L from African swine fever virus and BHRF1 from Epstein–Barr virus. We generated two sets of 8000 randomized BH3-like sequences from eight wild-type proapoptotic BH3 peptides. During this process, the four conserved hydrophobic residues and an Asp residue were retained at their respective positions, and all other positions were substituted randomly without any bias. We constructed 8000 structures each for A179L and BHRF1 in complex with BH3-like sequences. Histograms of interaction energies calculated between the peptide and the protein resulted in negatively skewed distributions. The BH3-like peptides with high helical propensities selected from the negative tail of the respective interaction energy distributions exhibited more favorable interactions with A179L and BHRF1, and they are rich in basic residues. Molecular dynamics studies and electrostatic potential maps further revealed that both acidic and basic residues favorably interact with A179L, while only basic residues have the most favorable interactions with BHRF1. As in mammalian homologues, the role of long-range interactions and nonhotspot residues has to be taken into account while designing specific BH3-mimetic inhibitors for vBcl-2 homologues.

Promotion of 11/9‐helical folding in α/β‐peptides containing β2‐homoalanine residue

AbstractThe 11/9‐helix is one of nontraditional helices available to α/β‐peptides with alternating residue types. Several β‐amino acid residues are known to promote 11/9‐helical folding with two types of intramolecular hydrogen bonds. Both β2‐amino acids and β3‐amino acids are common acyclic β‐amino acids that can be regarded as homologs of α‐amino acids with the same side chain group. However, β2‐amimo acids have not been widely used as much as β3‐amino acids partly because β2‐residue is believed to be not as strong as β3‐residue in promoting a distinct conformation. Here we report 11/9‐helical folding of α/β‐peptides that consist of L‐α‐alanine and (S)‐β2‐homoalanine with residue alternation. In addition, circular dichroism study reveals that β2‐homoalanine promotes 11/9‐helical folding more strongly than β3‐homoalanine in polar protic solvent conditions.

Interplay between membrane active host defense peptides and heme modulates their assemblies and in vitro activity

In the emerging era of antimicrobial resistance, the susceptibility to co-infections of patients suffering from either acquired or inherited hemolytic disorders can lead to dramatic increase in mortality rates. Closely related, heme liberated during hemolysis is one of the major sources of iron, which is vital for both host and invading microorganisms. While recent intensive research in the field has demonstrated that heme exerts diverse local effects including impairment of immune cells functions, it is almost completely unknown how it may compromise key molecules of our innate immune system, such as antimicrobial host defense peptides (HDPs). Since HDPs hold great promise as natural therapeutic agents against antibiotic-resistant microbes, understanding the effects that may modulate their action in microbial infection is crucial. Here we explore how hemin can interact directly with selected HDPs and influence their structure and membrane activity. It is revealed that induced helical folding, large assembly formation, and altered membrane activity is promoted by hemin. However, these effects showed variations depending mainly on peptide selectivity toward charged lipids, and the affinity of the peptide and hemin to lipid bilayers. Hemin-peptide complexes are sought to form semi-folded co-assemblies, which are present even with model membranes resembling mammalian or bacterial lipid compositions. In vitro cell-based toxicity assays supported that toxic effects of HDPs could be attenuated due to their assembly formation. These results are in line with our previous findings on peptide-lipid-small molecule systems suggesting that small molecules present in the complex in vivo milieu can regulate HDP function. Inversely, diverse effects of endogenous compounds could also be manipulated by HDPs.

Helical Structures of Nylon-Like Oligomers Consisting of 1,2-Diamine and 1,2-Dicarboxylic Acid Building Blocks Containing a Five-Membered Ring Constraint.

A series of nylon-like oligomers was synthesized, which consisted of alternating cyclic 1,2-diamine and 1,2-dicarboxylic acid building blocks with a five-membered ring constraint. The nylon 2 4 oligomers are symmetric and display helical structures similar to the β-peptide 12-helix with intramolecular 12-membered ring hydrogen bonds. The cyclopentane moiety allows each building block to promote 12-helical folding. In addition, a tartaric acid derivative with the acetonide moiety increases the solubility of oligomers in common organic solvents and promotes helical folding.

Folding and Intrinsic Disorder of the Receptor Tyrosine Kinase KIT Insert Domain Seen by Conventional Molecular Dynamics Simulations.

The kinase insert domain (KID) of RTK KIT is the key recruitment region for downstream signalling proteins. KID, studied by molecular dynamics simulations as a cleaved polypeptide and as a native domain fused to KIT, showed intrinsic disorder represented by a set of heterogeneous conformations. The accurate atomistic models showed that the helical fold of KID is mainly sequence dependent. However, the reduced fold of the native KID suggests that its folding is allosterically controlled by the kinase domain. The tertiary structure of KID represents a compact array of highly variable α- and 310-helices linked by flexible loops playing a principal role in the conformational diversity. The helically folded KID retains a collapsed globule-like shape due to non-covalent interactions associated in a ternary hydrophobic core. The free energy landscapes constructed from first principles—the size, the measure of the average distance between the conformations, the amount of helices and the solvent-accessible surface area—describe the KID disorder through a collection of minima (wells), providing a direct evaluation of conformational ensembles. We found that the cleaved KID simulated with restricted N- and C-ends better reproduces the native KID than the isolated polypeptide. We suggest that a cyclic, generic KID would be best suited for future studies of KID f post-transduction effects.

Mechanism of collagen folding propagation studied by Molecular Dynamics simulations.

Collagen forms a characteristic triple helical structure and plays a central role for stabilizing the extra-cellular matrix. After a C-terminal nucleus formation folding proceeds to form long triple-helical fibers. The molecular details of triple helix folding process is of central importance for an understanding of several human diseases associated with misfolded or unstable collagen fibrils. However, the folding propagation is too rapid to be studied by experimental high resolution techniques. We employed multiple Molecular Dynamics simulations starting from unfolded peptides with an already formed nucleus to successfully follow the folding propagation in atomic detail. The triple helix folding was found to propagate involving first two chains forming a short transient template. Secondly, three residues of the third chain fold on this template with an overall mean propagation of ~75 ns per unit. The formation of loops with multiples of the repeating unit was found as a characteristic misfolding event especially when starting from an unstable nucleus. Central Gly→Ala or Gly→Thr substitutions resulted in reduced stability and folding rates due to structural deformations interfering with folding propagation.

Defining the Energetic Basis for a Conformational Switch Mediating Ligand-Independent Activation of Mutant Estrogen Receptors in Breast Cancer.

Although most primary estrogen receptor (ER)-positive breast cancers respond well to endocrine therapies, many relapse later as metastatic disease due to endocrine therapy resistance. Over one third of these are associated with mutations in the ligand-binding domain (LBD) that activate the receptor independent of ligand. We have used an array of advanced computational techniques rooted in molecular dynamics simulations, in concert with and validated by experiments, to characterize the molecular mechanisms by which specific acquired somatic point mutations give rise to ER constitutive activation. By comparing structural and energetic features of constitutively active mutants and ligand-bound forms of ER-LBD with unliganded wild-type (WT) ER, we characterize a spring force originating from strain in the Helix 11-12 loop of WT-ER, opposing folding of Helix 12 into the active conformation and keeping WT-ER off and disordered, with the ligand-binding pocket open for rapid ligand binding. We quantify ways in which this spring force is abrogated by activating mutations that latch (Y537S) or relax (D538G) the folded form of the loop, enabling formation of the active conformation without ligand binding. We also identify a new ligand-mediated hydrogen-bonding network that stabilizes the active, ligand-bound conformation of WT-ER LBD, and similarly stabilizes the active conformation of the ER mutants in the hormone-free state. IMPLICATIONS: Our investigations provide deep insight into the energetic basis for the structural mechanisms of receptor activation through mutation, exemplified here with ER in endocrine-resistant metastatic breast cancers, with potential application to other dysregulated receptor signaling due to driver mutations.

Solvent Induced Helix Folding of Defined Indolenine Squaraine Oligomers.

A protecting group strategy was employed to synthesise a series of indolenine squaraine dye oligomers up to the nonamer. The longer oligomers show a distinct solvent dependence of the absorption spectra, that is, either a strong blue shift or a strong red shift of the lowest energy bands in the near infrared spectral region. This behaviour is explained by exciton coupling theory as being due to H‐ or J‐type coupling of transition moments. The H‐type coupling is a consequence of a helix folding in solvents with a small Hansen dispersity index. DOSY NMR, small angle neutron scattering (SANS), quantum chemical and force field calculations agree upon a helix structure with an unusually large pitch and open voids that are filled with solvent molecules, thereby forming a kind of clathrate. The thermodynamic parameters of the folding process were determined by temperature dependent optical absorption spectra.