Fast collective motions of backbone in transmembrane α helices are critical to water transfer of aquaporin.

Fast collective motions are widely present in biomolecules, but their functional relevance remains unclear. Herein, we reveal that fast collective motions of backbone are critical to the water transfer of aquaporin Z (AqpZ) by using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. A total of 212 residue site-specific dipolar order parameters and 158 15N spin relaxation rates of the backbone are measured by combining the 13C- and 1H-detected multidimensional ssNMR spectra. Analysis of these experimental data by theoretic models suggests that the small-amplitude (~10°) collective motions of the transmembrane α helices on the nanosecond-to-microsecond timescales are dominant for the dynamics of AqpZ. The MD simulations demonstrate that these collective motions are critical to the water transfer efficiency of AqpZ by facilitating the opening of the channel and accelerating the water-residue hydrogen bonds renewing in the selectivity filter region.

Insights on the G protein-coupled receptor helix 8 solution structure and orientation using a neurotensin receptor 1 peptide.

G-protein coupled receptors (GPCRs) are the largest class of membrane proteins encoded in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminal tail (Ctail). Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the Ctail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and Ctail (H8-Ctail) to investigate its structural stability, conformational dynamics, and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift-derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement NMR. Taken together, our studies reveal the H8-Ctail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.

Membrane-Active All-Hydrocarbon-Stapled α-Helical Amphiphilic Tat Peptides: Broad-Spectrum Antibacterial Activity and Low Incidence of Drug Resistance.

Multidrug resistance against conventional antibiotics has dramatically increased the difficulty of treatment and accelerated the need for novel antibacterial agents. The peptide Tat (47-57) is derived from the transactivating transcriptional activator of human immunodeficiency virus 1, which is well-known as a cell-penetrating peptide in mammalian cells. However, it is also reported that the Tat peptide (47-57) has antifungal activity. In this study, a series of membrane-active hydrocarbon-stapled α-helical amphiphilic peptides were synthesized and evaluated as antibacterial agents against Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. The impact of hydrocarbon staple, the position of aromatic amino acid residue in the hydrophobic face, the various types of aromatic amino acids, and the hydrophobicity on bioactivity were also investigated and discussed in this study. Among those synthesized peptides, analogues P3 and P10 bearing a l-2-naphthylalanine (Φ) residue at the first position and a Tyr residue at the eighth position demonstrated the highest antimicrobial activity and negligible hemolytic toxicity. Notably, P3 and P10 showed obviously enhanced antimicrobial activity against multidrug-resistant bacteria, low drug resistance, high cell selectivity, extended half-life in plasma, and excellent performance against biofilm. The antibacterial mechanisms of P3 and P10 were also preliminarily investigated in this effort. In conclusion, P3 and P10 are promising antimicrobial alternatives for the treatment of the antimicrobial-resistance crisis.

Staphylococcal peroxidase inhibitor (SPIN): Residue-level investigation of the helical bundle domain.

Myeloperoxidase is a critical component of the antibacterial arsenal of neutrophils, whereby it consumes H2O2 as an oxidant to convert halogen and pseudohalogen anions into cytotoxic hypohalous acids. Following phagocytosis by neutrophils, the human pathogen Staphylococcus aureus secretes a potent myeloperoxidase inhibitory protein, called SPIN, as part of its immune evasion repertoire. The matured S. aureus SPIN polypeptide consists of only 73 residues yet contains two functional domains: whereas the 60 residue C-terminal helical bundle domain is responsible for MPO binding, the 13 residue N-terminal domain is required to inhibit MPO. Previous studies have informed understanding of the SPIN N-terminal domain, but comparatively little is known about the helical domain insofar as the contribution of individual residues is concerned. To address this limitation, we carried out a residue-level structure/function investigation on the helical bundle domain of S. aureus SPIN. Using sequence conservation and existing structures of SPIN bound to human MPO as a guide, we selected residues L49, E50, H51, E52, Y55, and Y75 for interrogation by site-directed mutagenesis. We found that loss of L49 or E52 reduced SPIN activity by roughly an order of magnitude, but that loss of Y55 or H51 caused progressively greater loss of inhibitory potency. Direct binding studies by SPR showed that loss of inhibitory potency in these SPIN mutants resulted from a diminished initial interaction between the inhibitor and MPO. Together, our studies provide new insights into the structure/function relationships of SPIN and identify positions Y55 and H51 as critical determinants of SPIN function.

Architectonic principles of polyproline II helix bundle protein domains.

Glycine rich polyproline II helix assemblies are an emerging class of natural domains found in several proteins with different functions and diverse origins. The distinct properties of these domains relative to those composed of α-helices and ß-sheets could make glycine-rich polyproline II helix assemblies a useful building block for protein design. Whereas the high population of polyproline II conformers in disordered state ensembles could facilitate glycine-rich polyproline II helix folding, the architectonic bases of these structures are not well known. Here, we compare and analyze their structures to uncover common features. These protein domains are found to be highly tolerant of distinct flanking sequences. This speaks to the robustness of this fold and strongly suggests that glycine rich polyproline II assemblies could be grafted with other protein domains to engineer new structures and functions. These domains are also well packed with few or no cavities. Moreover, a significant trend towards antiparallel helix configuration is observed in all these domains and could provide stabilizing interactions among macrodipoles. Finally, extensive networks of Cα-H···OC hydrogen bonds are detected in these domains. Despite their diverse evolutionary origins and activities, glycine-rich polyproline II helix assemblies share architectonic features which could help design novel proteins.

Enzyme-Triggered Polyelectrolyte Complex for Responsive Delivery of α-Helical Polypeptides to Optimize Antibacterial Therapy.

Responsive nanomaterials hold significant promise in the treatment of bacterial infections by recognizing internal or external stimuli to achieve stimuli-responsive behavior. In this study, we present an enzyme-responsive polyelectrolyte complex micelles (PTPMN) with α-helical cationic polypeptide as a coacervate-core for the treatment of Escherichia coli (E. coli) infection. The complex was constructed through electrostatic interaction between cationic poly(glutamic acid) derivatives and phosphorylation-modified poly(ethylene glycol)-b-poly(tyrosine) (PEG-b-PPTyr) by directly dissolving them in aqueous solution. The cationic polypeptide adopted α-helical structure and demonstrated excellent broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with a minimum inhibitory concentration (MIC) as low as 12.5 µg mL-1 against E. coli. By complexing with anionic PEG-b-PPTyr, the obtained complex formed ß-sheet structures and exhibited good biocompatibility and low hemolysis. When incubated in a bacterial environment, the complex cleaved its phosphate groups triggered by phosphatases secreted by bacteria, exposing the highly α-helical conformation and restoring its effective bactericidal ability. In vivo experiments confirmed accelerated healing in E. coli-infected wounds.

Structural insights into the functional mechanism of the ubiquitin ligase E6AP.

E6AP dysfunction is associated with Angelman syndrome and Autism spectrum disorder. Additionally, the host E6AP is hijacked by the high-risk HPV E6 to aberrantly ubiquitinate the tumor suppressor p53, which is linked with development of multiple types of cancer, including most cervical cancers. Here we show that E6AP and the E6AP/E6 complex exist, respectively, as a monomer and a dimer of the E6AP/E6 protomer. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6. The extended α1-helices of the dimer intersect symmetrically and contribute to the dimerization. The two protomers sway around the crossed region of the two α1-helices to promote the attachment and detachment of substrates to the catalytic C-lobe of E6AP, thus facilitating ubiquitin transfer. These findings, complemented by mutagenesis analysis, suggest that the α1-helix, through conformational transformations, controls the transition between the inactive monomer and the active dimer of E6AP.

Antimicrobial Activities of α-Helix and ß-Sheet Peptides against the Major Bovine Respiratory Disease Agent, Mannheimia haemolytica.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle raised in North America. At the feedlot, cattle are subject to metaphylactic treatment with macrolides to prevent BRD, a practice that may promote antimicrobial resistance and has resulted in an urgent need for novel strategies. Mannheimia haemolytica is one of the major bacterial agents of BRD. The inhibitory effects of two amphipathic, α-helical (PRW4, WRL3) and one ß-sheet (WK2) antimicrobial peptides were evaluated against multidrug-resistant (MDR) M. haemolytica isolated from Alberta feedlots. WK2 was not cytotoxic against bovine turbinate (BT) cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. All three peptides inhibited M. haemolytica, with WK2 being the most efficacious against multiple isolates. At 8-16 µg/mL, WK2 was bactericidal against Mh 330 in broth, and at 32 µg/mL in the presence of BT cells, it reduced the population by 3 logs CFU/mL without causing cytotoxic effects. The membrane integrity of Mh 330 was examined using NPN (1-N-phenylnaphthylamine) and ONPG (o-Nitrophenyl ß-D-galactopyranoside), with both the inner and outer membranes being compromised. Thus, WK2 may be a viable alternative to the use of macrolides as part of BRD prevention and treatment strategies.

Effects of Proline on Internal Friction in Simulated Folding Dynamics of Several Alanine-Based α-Helical Peptides.

We have studied in silico the effect of proline, a model cosolvent, on local and global friction coefficients in (un)folding of several typical alanine-based α-helical peptides. Local friction is related to dwell times of a single, ensemble-averaged hydrogen bond (HB) within each peptide. Global friction is related to energy dissipated in a series of configurational changes of each peptide experienced by increasing the number of HBs during folding. Both of these approaches are important in relation to future atomic force microscopic-based measurements of internal friction via force-clamp single-molecule force spectroscopy. Molecular dynamics (MD) simulations for six peptides, namely, ALA5, ALA8, ALA15, ALA21, (AAQAA)3, and H2N-GN(AAQAA)2G-COONH2, have been conducted at 2 and 5 M proline solutions in water. Using previously obtained MD data for these peptides in pure water as well as upgraded theoretical models, we obtained variations of local and global internal friction coefficients as a function of solution viscosity. The results showed the substantial role of proline in stabilizing the folded state and slowing the overall folding dynamics. Consequently, larger friction coefficients were obtained at larger viscosities. The local and global internal friction, i.e., respective, friction coefficients approximated to zero viscosity, was also obtained. The evolution of friction coefficients with viscosity was weakly dependent on the number of concurrent folding pathways but was rather dominated by a stabilizing effect of proline on the folded states. Obtained values of local and global internal friction showed qualitatively similar results and a clear dependency on the structure of the studied peptide.

Helical Content Correlations and Hydration Structures of the Folding Ensemble of the B Domain of Protein A.

The B domain of protein A (BdpA), a small three-helix bundle, folds on a time scale of a few microseconds with heterogeneous native and unfolded states. It is widely used as a model for understanding protein folding mechanisms. In this work, we use structure-based models (SBMs) and atomistic simulations to comprehensively investigate how BdpA folding is associated with the formation of its secondary structure. The energy landscape visualization method (ELViM) was used to characterize the pathways that connect the folded and unfolded states of BdpA as well as the sets of structures displaying specific ellipticity patterns. We show that the native state conformational diversity is due mainly to the conformational variability of helix I. Helices I, II, and III occur in a weakly correlated manner, with Spearman's rank correlation coefficients of 0.1539 (I and II), 0.1259 (I and III), and 0.2561 (II and III). These results, therefore, suggest the highest cooperativity between helices II and III. Our results allow the clustering of partially folded structures of folding of the B domain of protein A on the basis of its secondary structure, paving the way to an understanding of environmental factors in the relative stability of the basins of the folding ensemble, which are illustrated by the structural dependency of the protein hydration structures, as computed with minimum-distance distribution functions.

Acyl Capping Group Identity Effects on α-Helicity: On the Importance of Amide·Water Hydrogen Bonds to α-Helix Stability.

Acyl capping groups stabilize α-helices relative to free N-termini by providing one additional C═Oi···Hi+4-N hydrogen bond. The electronic properties of acyl capping groups might also directly modulate α-helix stability: electron-rich N-terminal acyl groups could stabilize the α-helix by strengthening both i/i + 4 hydrogen bonds and i/i + 1 n → π* interactions. This hypothesis was tested in peptides X-AKAAAAKAAAAKAAGY-NH2, where X = different acyl groups. Surprisingly, the most electron-rich acyl groups (pivaloyl and iso-butyryl) strongly destabilized the α-helix. Moreover, the formyl group induced nearly identical α-helicity to that of the acetyl group, despite being a weaker electron donor for hydrogen bonds and for n → π* interactions. Other acyl groups exhibited intermediate α-helicity. These results indicate that the electronic properties of the acyl carbonyl do not directly determine the α-helicity in peptides in water. In order to understand these effects, DFT calculations were conducted on α-helical peptides. Using implicit solvation, α-helix stability correlated with acyl group electronics, with the pivaloyl group exhibiting closer hydrogen bonds and n → π* interactions, in contrast to the experimental results. However, DFT and MD calculations with explicit water solvation revealed that hydrogen bonding to water was impacted by the sterics of the acyl capping group. Formyl capping groups exhibited the closest water-amide hydrogen bonds, while pivaloyl groups exhibited the longest. In α-helices in the PDB, the highest frequency of close amide-water hydrogen bonds is observed when the N-cap residue is Gly. The combination of experimental and computational results indicates that solvation (hydrogen bonding of water) to the N-terminal amide groups is a central determinant of α-helix stability.

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 ß-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 ß-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.

Molecular dynamics simulations suggest the potential toxicity of fluorinated graphene to HP35 protein via unfolding the α-helix structure.

Fluorinated graphene, a two-dimensional nanomaterial composed of three atomic layers, a central carbon layer sandwiched between two layers of fluorine atoms, has attracted considerable attention across various fields, particularly for its potential use in biomedical applications. Nonetheless, scant effort has been devoted to assessing the potential toxicological implications of this nanomaterial. In this study, we scrutinize the potential impact of fluorinated graphene on a protein model, HP35 by utilizing extensive molecular dynamics (MD) simulation methods. Our MD results elucidate that upon adsorption to the nanomaterial, HP35 undergoes a denaturation process initiated by the unraveling of the second helix of the protein and the loss of the proteins hydrophobic core. In detail, substantial alterations in various structural features of HP35 ensue, including alterations in hydrogen bonding, Q value, and RMSD. Subsequent analyses underscore that hydrophobic and van der Waals interactions (predominant), alongside electrostatic energy (subordinate), exert influence over the adsorption of HP35 on the fluorinated graphene surface. Mechanistic scrutiny attests that the unrestrained lateral mobility of HP35 on the fluorinated graphene nanomaterial primarily causes the exposure of HP35's hydrophobic core, resulting in the eventual structural denaturation of HP35. A trend in the features of 2D nanostructures is proposed that may facilitate the denaturation process. Our findings not only substantiate the potential toxicity of fluorinated graphene but also unveil the underlying molecular mechanism, which thereby holds significance for the prospective utilization of such nanomaterials in the field of biomedicine.

Modification of soy protein isolate and pea protein isolate by high voltage dielectric barrier discharge (DBD) atmospheric cold plasma: Comparative study on structural, rheological and techno-functional characteristics.

The modification in structural, rheological, and techno-functional characteristics of soy and pea protein isolates (SPI and PPI) due to dielectric barrier discharge cold plasma (DBD-CP) were assessed. The increased carbonyl groups in both samples with cold plasma (CP) treatment led to a reduction in free sulfhydryl groups. Moreover, protein solubility of treated proteins exhibited significant improvements, reaching up to 59.07 % and 41.4 % for SPI and PPI, respectively, at 30 kV for 8 min. Rheological analyses indicated that storage modulus (G') was greater than loss modulus (G″) for CP-treated protein gels. Furthermore, in vitro protein digestibility of SPI exhibited a remarkable improvement (4.78 %) at 30 kV for 6 min compared to PPI (3.23 %). Spectroscopic analyses, including circular dichroism and Fourier Transform-Raman, indicated partial breakdown and loss of α-helix structure in both samples, leading to the aggregation of proteins. Thus, DBD-CP induces reactive oxygen species-mediated oxidation, modifying the secondary and tertiary structures of samples.

Unraveling the binding mechanism between soybean protein isolate and selected bioactive compounds.

The present study was aimed to investigate the interactions between soybean protein isolate (SPI) with resveratrol (RESV) and lutein (LUT). The binding forces, molecular interactions and functional properties were explored by multi-spectroscopic analysis, molecular docking and functional property indexes between SPI and RESV/LUT. The RESV/LUT quenched SPI chromophore residues with static mechanism and the endothermic reaction. The SPI- RESV/LUT complexes were formed through hydrogen bond, electrostatic and hydrophobic interactions. Molecular docking confirmed van-der-Waals force as one of the important forces. The interaction of RESV/LUT led to SPI's secondary structure alterations with a decrease in α-helix and random coil and an increase in ß-sheet and ß-turns. RESV/LUT developed foaming and emulsifying properties of SPI and showed a significant decrease of the surface hydrophobicity with RESV/LUT concentrations increase attributed to SPI's partial unfolding. Our study exposed molecular mechanisms and confirmations to understand the interactions in protein- RESV/LUT complexes for protein industrial base promotion.

Rational Design of TDP-43 Derived α-Helical Peptide Inhibitors: An In Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders.

TDP-43, an essential RNA/DNA-binding protein, is central to the pathology of neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia. Pathological mislocalization and aggregation of TDP-43 disrupt RNA splicing, mRNA stability, and mRNA transport, thereby impairing neuronal function and survival. The formation of amyloid-like TDP-43 filaments is largely facilitated by the destabilization of an α-helical segment within the disordered C-terminal region. In this study, we hypothesized that preventing the destabilization of the α-helical domain could potentially halt the growth of these pathological filaments. To explore this, we utilized a range of in silico techniques to design and evaluate peptide-based therapeutics that bind to pathological TDP-43 amyloid-like filament crystal structures and resist ß sheet conversion. Our computational approaches, including biophysical and secondary structure property prediction, molecular docking, 3D structure prediction, and molecular dynamics simulations, were used to assess the structure, stability, and binding affinity of these peptides in relation to pathological TDP-43 filaments. The results of our in silico analyses identified a selection of promising peptides which displayed a stable α-helical structure, exhibited an increased number of intramolecular hydrogen bonds within the helical domain, and demonstrated high binding affinities for pathological TDP-43 amyloid-like filaments. Molecular dynamics simulations provided further support for the structural and thermodynamic stability of these peptides, as they exhibited lower root-mean-square deviation and more favorable free energy landscapes over 300 ns. These findings establish α-helical propensity peptides as potential lead molecules for the development of novel therapeutics against TDP-43 aggregation. This structure-based computational approach for the rational design of peptide inhibitors opens a new direction in the search for effective interventions for ALS, FTD, and other related neurodegenerative diseases. The peptides identified as the most promising candidates in this study are currently subject to further testing and validation through both in vitro and in vivo experiments.

Folding of Class IIa HDAC Derived Peptides into α-helices Upon Binding to Myocyte Enhancer Factor-2 in Complex with DNA.

Interaction of transcription factor myocyte enhancer factor-2 (MEF2) family members with class IIa histone deacetylases (HDACs) has been implicated in a wide variety of diseases. Though considerable knowledge on this topic has been accumulated over the years, a high resolution and detailed analysis of the binding mode of multiple class IIa HDAC derived peptides with MEF2D is still lacking. To fulfil this gap, we report here the crystal structure of MEF2D in complex with double strand DNA and four different class IIa HDAC derived peptides, namely HDAC4, HDAC5, HDAC7 and HDAC9. All class IIa HDAC derived peptides form extended amphipathic α-helix structures that fit snugly in the hydrophobic groove of MEF2D domain. Binding mode of class IIa HDAC derived peptides to MEF2D is very similar and occur primarily through nonpolar interactions mediated by highly conserved branched hydrophobic amino acids. Further studies revealed that class IIa HDAC derived peptides are unstructured in solution and appear to adopt a folded α-helix structure only upon binding to MEF2D. Comparison of our peptide-protein complexes with previously characterized structures of MEF2 bound to different co-activators and co-repressors, highlighted both differences and similarities, and revealed the adaptability of MEF2 in protein-protein interactions. The elucidation of the three-dimensional structure of MEF2D in complex with multiple class IIa HDAC derived peptides provide not only a better understanding of the molecular basis of their interactions but also have implications for the development of novel antagonist.

Identification of a conserved α-helical domain at the N terminus of human DNA methyltransferase 1.

In vertebrates, DNA methyltransferase 1 (DNMT1) contributes to preserving DNA methylation patterns, ensuring the stability and heritability of epigenetic marks important for gene expression regulation and the maintenance of cellular identity. Previous structural studies have elucidated the catalytic mechanism of DNMT1 and its specific recognition of hemimethylated DNA. Here, using solution nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, we demonstrate that the N-terminal region of human DNMT1, while flexible, encompasses a conserved globular domain with a novel α-helical bundle-like fold. This work expands our understanding of the structure and dynamics of DNMT1 and provides a structural framework for future functional studies in relation with this new domain.

The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase.

XK-related 8 (XKR8), in complex with the transmembrane glycoprotein basigin, functions as a phospholipid scramblase activated by the caspase-mediated cleavage or phosphorylation of its C-terminal tail. It carries a putative phospholipid translocation path of multiple hydrophobic and charged residues in the transmembrane region. It also has a crucial tryptophan at the exoplasmic end of the path that regulates its scrambling activity. We herein investigated the tertiary structure of the human XKR8-basigin complex embedded in lipid nanodiscs at an overall resolution of 3.66 Å. We found that the C-terminal tail engaged in intricate polar and van der Waals interactions with a groove at the cytoplasmic surface of XKR8. These interactions maintained the inactive state of XKR8. Point mutations to disrupt these interactions strongly enhanced the scrambling activity of XKR8, suggesting that the activation of XKR8 is mediated by releasing the C-terminal tail from the cytoplasmic groove. We speculate that the cytoplasmic tail region of XKR8 functions as a plug to prevent the scrambling of phospholipids.

Folded Alpha Helical Putative New Proteins from Apilactobacillus kunkeei.

The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0-3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.