Single Alpha-helices Research Articles

Fish antifreeze protein origin in sculpins by frameshifting within a duplicated housekeeping gene.

Antifreeze proteins (AFPs) are found in a variety of marine cold-water fishes where they prevent freezing by binding to nascent ice crystals. Their diversity (types I, II, III and antifreeze glycoproteins), as well as their scattered taxonomic distribution hint at their complex evolutionary history. In particular, type I AFPs appear to have arisen in response to the Late Cenozoic Ice Age that began ~ 34 million years ago via convergence in four different groups of fish that diverged from lineages lacking this AFP. The progenitor of the alanine-rich α-helical type I AFPs of sculpins has now been identified as lunapark, an integral membrane protein of the endoplasmic reticulum. Following gene duplication and loss of all but three of the 15 exons, the final exon, which encoded a glutamate- and glutamine-rich segment, was converted to an alanine-rich sequence by a combination of frameshifting and mutation. Subsequent gene duplications produced numerous isoforms falling into four distinct groups. The origin of the flounder type I AFP is quite different. Here, a small segment from the original antiviral protein gene was amplified and the rest of the coding sequence was lost, while the gene structure was largely retained. The independent origins of type I AFPs with up to 83% sequence identity in flounder and sculpin demonstrate strong convergent selection at the level of protein sequence for alanine-rich single alpha helices that bind to ice. Recent acquisition of these AFPs has allowed sculpins to occupy icy seawater niches with reduced competition and predation from other teleost species.

Uncovering the Contributions of Charge Regulation to the Stability of Single Alpha Helices.

The single alpha helix (SAH) is a recurring motif in biology. The consensus sequence has a di-block architecture that includes repeats of four consecutive glutamate residues followed by four consecutive lysine residues. Measurements show that the overall helicity of sequences with consensus E4 K4 repeats is insensitive to a wide range of pH values. Here, we use the recently introduced q-canonical ensemble, which allows us to decouple measurements of charge state and conformation, to explain the observed insensitivity of SAH helicity to pH. We couple the outputs from separate measurements of charge and conformation with atomistic simulations to derive residue-specific quantifications of preferences for being in an alpha helix and for the ionizable residues to be charged vs. uncharged. We find a clear preference for accommodating uncharged Glu residues within internal positions of SAH-forming sequences. The stabilities of alpha helical conformations increase with the number of E4 K4 repeats and so do the numbers of accessible charge states that are compatible with forming conformations of high helical content. There is conformational buffering whereby charge state heterogeneity buffers against large-scale conformational changes thus making the overall helicity insensitive to large changes in pH. Further, the results clearly argue against a single, rod-like alpha helical conformation being the only or even dominant conformation in the ensembles of so-called SAH sequences.

Charge state heterogeneity in single alpha helices helps maintain helicity over a large range of pH values

Single Alpha Helices are repetitive, highly charged polyampholytes that exhibit a stable helical structure, despite having many characteristics of intrinsically disordered proteins. Previous studies have proposed that i, i+4 salt-bridges may be a key to the stabilization of the helical structure. However, measurements have also shown that SAHs can be stable over a large range of pH values, calling into question whether salt-bridges persist as stabilizing factors away from neutral pH values, and if they are the only contributors at neutral pH.

The Sensitivity of the Pair-Angle Distribution Function to Protein Structure

The continued development of X-ray free-electron lasers and serial crystallography techniques has opened up new experimental frontiers. Nanoscale dynamical processes such as crystal growth can now be probed at unprecedented time and spatial resolutions. Pair-angle distribution function (PADF) analysis is a correlation-based technique that has the potential to extend the limits of current serial crystallography experiments, by relaxing the requirements for crystal order, size and number density per exposure. However, unlike traditional crystallographic methods, the PADF technique does not recover the electron density directly. Instead it encodes substantial information about local three-dimensional structure in the form of three- and four-body correlations. It is not yet known how protein structure maps into the many-body PADF correlations. In this paper, we explore the relationship between the PADF and protein conformation. We calculate correlations in reciprocal and real space for model systems exhibiting increasing degrees of order and secondary structural complexity, from disordered polypeptides, single alpha helices, helix bundles and finally a folded 100 kilodalton protein. These models systems inform us about the distinctive angular correlations generated by bonding, polypeptide chains, secondary structure and tertiary structure. They further indicate the potential to use angular correlations as a sensitive measure of conformation change that is complementary to existing structural analysis techniques.

Quantifying the Effects of Charge Regulation on Disorder-Order Transitions of Highly Charged Protein Sequences

Charge regulation, which refers to the modulation of charge states of ionizable residues due to shifted pKa values, is an important albeit often overlooked determinant of sequence-ensemble and sequence-structure relationships of intrinsically disordered and intrinsically ordered proteins. We have developed complimentary computational and experimental approaches that account for the complete ensemble of charge states for a given sequence. This combined approach enables the computational calculation of conformational ensembles as a function of pH, and experimental measurement of charge states as a function of pH, allowing us to quantify the effects of charge regulation on archetypal sequences that are predicted to be intrinsically disordered. Here, we present results from a joint experimental and computational analysis of the effects of charge regulation on block-copolypeptides composed entirely of acidic and basic residues, (E4K4)n. These sequences form stable single alpha helices, although they are erroneously predicted to form disordered ensembles if one assumes fixed charge states for the ionizable residues. We employ a novel capillary electrophoresis method for measuring charge as a function of pH. These data, combined with potentiometric analysis, show that glutamate residues have highly shifted pKa values when compared to those of model compounds. This preferential neutralization of glutamate residues that are internal to the block co-polypeptide sequence engenders alpha helix formation by lowering the free energy of desolvation and enabling the favorable propagation of the alpha helix. These findings are readily explained using simulations based on the recently developed q-canonical Monte Carlo sampling method. Our results suggest that sequence-specific charge regulation can engender large-scale conformational transitions and even underlie the driving forces for phase separation. Further, our results highlight the need for refining disorder predictors based on an improved understanding of context dependent charge regulation effects.

Glutamine Side-Chain to Main Chain Hydrogen Bonds Can be used to Design Single Alpha-Helices that are Stable at Room Temperature

The genetic instability of polyCAG repeats is the cause of polyglutamine (polyQ) tract expansions, which is a potential molecular basis of a group of disorders known as polyQ diseases. PolyQ tracts are common sequences rich in glutamine residues mainly found in intrinsically disordered regions of proteins related to transcription. Their expansion has been hypothesized to induce a conformational change affecting their solubility that results in the formation of fibrillar aggregates in specific tissues of patients affected by polyQ diseases. With most in vitro studies reporting a collapsed random coil arrangement with no major conformational shifts upon expansion, recent studies have shown a certain propensity to form helices that increases with tract length. In physiological conditions, this effect may be enhanced by the interactions with binding partners as well as by partitioning proteins bearing polyQ tracts in phase-separated protein condensates. Such an environment can favour the formation of the side-chain (i) to main-chain (i-4) hydrogen bonds that we recently reported as a stabilizing factor of polyQ helices (Escobedo, A. et al. Nat. Commun. 10, 2034 (2019)). We here show that this is a common feature of polyQ tracts, unveil bulkiness and hydrophobicity of the accepting residue type as the main factors affecting the strength of the interaction, and exploit this knowledge to generate uncharged single α-helices that are stable at room temperature and therefore suitable for protein and biomaterials design.

Detection of single alpha-helices in large protein sequence sets using hardware acceleration.

Single alpha-helices (SAHs) are increasingly recognized as important structural and functional elements of proteins. Comprehensive identification of SAH segments in large protein datasets was largely hindered by the slow speed of the most restrictive prediction tool for their identification, FT_CHARGE on common hardware. We have previously implemented an FPGA-based version of this tool allowing fast analysis of a large number of sequences. Using this implementation, we have set up of a semi-automated pipeline capable of analyzing full UniProt releases in reasonable time and compiling monthly updates of a comprehensive database of SAH segments. Releases of this database, denoted CSAHDB, is available on the CSAHserver 2 website at csahserver.itk.ppke.hu. An overview of human SAH-containing sequences combined with a literature survey suggests specific roles of SAH segments in proteins involved in RNA-based regulation processes as well as cytoskeletal proteins, a number of which is also linked to the development and function of synapses.

Consensus Prediction of Charged Single Alpha-Helices with CSAHserver.

Charged single alpha-helices (CSAHs) constitute a rare structural motif. CSAH is characterized by a high density of regularly alternating residues with positively and negatively charged side chains. Such segments exhibit unique structural properties; however, there are only a handful of proteins where its existence is experimentally verified. Therefore, establishing a pipeline that is capable of predicting the presence of CSAH segments with a low false positive rate is of considerable importance. Here we describe a consensus-based approach that relies on two conceptually different CSAH detection methods and a final filter based on the estimated helix-forming capabilities of the segments. This pipeline was shown to be capable of identifying previously uncharacterized CSAH segments that could be verified experimentally. The method is available as a web server at http://csahserver.itk.ppke.hu and also a downloadable standalone program suitable to scan larger sequence collections.

Charge Patterned Sequences form Helical Structures through Charge Neutralization

Many proteins with repeating blocks of oppositely charged residues form long single alpha helices (SAHs). The consensus sequence is a block of four Glu residues followed by a block of four Lys or Arg residues, (Glu4(Lys/Arg)4)n. The current working hypothesis is that SAHs are stabilized by i:i+4 salt bridges between opposite charges in consecutive helical turns. We test the merits of this hypothesis to understand the sequence-encoded preference for SAHs and the logic behind the failure of certain atomistic simulations in anticipating the preference for stable SAHs.In simulations with fixed charges the favorable free energy of solvation of charged residues and the associated loss of sidechain entropy hinders the formation of SAHs. We proposed that alterations to charge states induced by sequence context might play an important role in stabilizing SAHs. We tested this hypothesis using a (Glu4Lys4)n repeat protein and a simulation strategy that permits the substitution of charged residues with neutralized protonated or deprotonated variants of Glu / Lys. Our results predict that stable SAH structures derive from the neutralization of approximately half the Glu residues. These findings explain experimental observations and also provide a coherent rationale for the failure of simulations based on fixed charge models. Large-scale sequence analysis reveals that naturally occurring sequences often include “defects” in charge patterns such as Gln or Ala substitutions. This sequence-encoded incorporation of uncharged residues combined with neutralization of charged residues might tilt the balance toward alpha helical conformations.Our results highlight the need for developing robust methodologies for constant pH simulations that can be applied to sequences with high charge contents. They also highlight the need for generalizing bioinformatics predictors to account for sequence-encoded charge regulation that might influence disorder predictions for sequences with high fractions of charged residues.

Charged single alpha-helices in proteomes revealed by a consensus prediction approach

Charged single α-helices (CSAHs) constitute a recently recognized protein structural motif. Its presence and role is characterized in only a few proteins. To explore its general features, a comprehensive study is necessary. We have set up a consensus prediction method available as a web service (at http://csahserver.chem.elte.hu) and downloadable scripts capable of predicting CSAHs from protein sequences. Using our method, we have performed a comprehensive search on the UniProt database. We found that the motif is very rare but seems abundant in proteins involved in symbiosis and RNA binding/processing. Although there are related proteins with CSAH segments, the motif shows no deep conservation in protein families. We conclude that CSAH-containing proteins, although rare, are involved in many key biological processes. Their conservation pattern and prevalence in symbiosis-associated proteins suggest that they might be subjects of relatively rapid molecular evolution and thus can contribute to the emergence of novel functions.

Combining Single Molecule Optical Trapping and Small Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein Alpha-Helix

A relatively unknown protein structure motif forms stable isolated single alpha-helices, termed ER/K alpha-helices, in a wide variety of proteins and has been shown to be essential for the function of some molecular motors. The stability of the ER/K alpha -helix arises from the charge-charge interactions between its glutamic acid (E) and Arginine (R) or Lysine (K) side chains. The flexibility of the ER/K alpha-helix determines whether it behaves as a force-transducer, rigid spacer or flexible linker in proteins. The ER/K alpha-helix spans long distances with relatively few amino acid residues, has known salt and temperature sensitivity, and can be expressed in E. coli, making it an important tool in engineering proteins, provided its mechanical properties are clearly established. We have quantified the flexibility of the ER/K alpha-helix in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small angle x-ray scattering (SAXS), combined with Montecarlo simulations to demonstrate that the ER/K alpha-helix behaves as a worm-like-chain with persistence length of ∼ 15 nm. This persistence length is dependent on the relative content of R and K residues in the ER/K alpha-helix. Knowledge of the persistence length enables us to define its function as a rigid spacer in a translation initiation factor, as a force-transducer in the mechanoenzyme myosin VI, and as a flexible spacer in the Kelch motif containing protein.

Charged single α‐helix: A versatile protein structural motif

A few highly charged natural peptide sequences were recently suggested to form stable alpha-helical structures in water. In this article we show that these sequences represent a novel structural motif called "charged single alpha-helix" (CSAH). To obtain reliable candidate CSAH motifs, we developed two conceptually different computational methods capable of scanning large databases: SCAN4CSAH is based on sequence features characteristic for salt bridge stabilized single alpha-helices, whereas FT_CHARGE applies Fourier transformation to charges along sequences. Using the consensus of the two approaches, a remarkable number of proteins were found to contain putative CSAH domains. Recombinant fragments (50-60 residues) corresponding to selected hits obtained by both methods (myosin 6, Golgi resident protein GCP60, and M4K4 protein kinase) were produced and shown by circular dichroism spectroscopy to adopt largely alpha-helical structure in water. CSAH segments differ substantially both from coiled-coil and intrinsically disordered proteins, despite the fact that current prediction methods recognize them as either or both. Analysis of the proteins containing CSAH motif revealed possible functional roles of the corresponding segments. The suggested main functional features include the formation of relatively rigid spacer/connector segments between functional domains as in caldesmon, extension of the lever arm in myosin motors and mediation of transient interactions by promoting dimerization in a range of proteins.

Superelasticity, energy dissipation and strain hardening of vimentin coiled-coil intermediate filaments: atomistic and continuum studies

Vimentin coiled-coil alpha-helical dimers are elementary protein building blocks of intermediate filaments, an important component of the cell’s cytoskeleton that has been shown to control the large-deformation behavior of eukaryotic cells. Here we use a combination of atomistic simulation and continuum theory to model tensile and bending deformation of single alpha-helices as well as coiled-coil double helices of the 2B segment of the vimentin dimer. We find that vimentin dimers can be extended to tensile strains up to 100% at forces below 50 pN, until strain hardening sets in with rapidly rising forces, approaching 8 nN at 200% strain. We systematically explore the differences between single alpha-helical structures and coiled-coil superhelical structures. Based on atomistic simulation, we discover a transition in deformation mechanism under varying pulling rates, resulting in different strength criteria for the unfolding force. Based on an extension of Bell’s theory that describes the dependence of the mechanical unfolding force on the pulling rate, we develop a fully atomistically informed continuum model of the mechanical properties of vimentin coiled-coils that is capable of predicting its nanomechanical behavior over a wide range of deformation rates that include experimental conditions. This model enables us to describe the mechanics of cyclic stretching experiments, suggesting a hysteresis in the force–strain response, leading to energy dissipation as the protein undergoes repeated tensile loading. We find that the dissipated energy increases continuously with increasing pulling rate. Our atomistic and continuum results help to interpret experimental studies that have provided evidence for the significnificance of vimentin intermediate filaments for the large-deformation regime of eukaryotic cells. We conclude that vimentin dimers are superelastic, highly dissipative protein assemblies.

Hierarchical nanomechanics of vimentin alpha helical coiled-coil proteins

AbstractCoiled-coil alpha-helical dimers are the elementary building blocks of intermediate filaments (IFs), an important component of the cell's cytoskeleton. Therefore, IFs play a leading role in the mechanical integrity of the cells. Here we use atomistic simulation to carry out tensile tests on coiled-coils as well as on single alpha-helices of the 2B segment of the vimentin dimer that has been shown to control the large-deformation behavior of cells. We compare the characteristic force-strain curves of both structures and suggest explanations for the differences on this fundamental level of hierarchical assembly. We further systematically explore the strain rate dependence of the mechanical properties of the vimentin coiled-coil protein. We develop a simple continuum model capable of reproducing the atomistic modeling results. The model enables us to extrapolate to much lower deformation rates approaching those used in experiment.

Refining the Structure of the Halobacterium salinarum Flagellar Filament Using the Iterative Helical Real Space Reconstruction Method: Insights into Polymorphism

The eubacterial flagellar filament is an external, self-assembling, helical polymer approximately 220 A in diameter constructed from a highly conserved monomer, flagellin, which polymerizes externally at the distal end. The archaeal filament is only approximately 100 A in diameter, assembles at the proximal end and is constructed from different, glycosylated flagellins. Although the phenomenology of swimming is similar to that of eubacteria, the symmetry of the archebacterial filament is entirely different. Here, we extend our previous study on the flagellar coiled filament structure of strain R1M1 of Halobacterium salinarum. We use strain M175 of H.salinarum, which forms poly-flagellar bundles at high yield which, under conditions of relatively low ionic-strength (0.8 M versus 5 M) and low pH ( approximately 2.5 versus approximately 6.8), form straight filaments. We demonstrated previously that a single-particle approach to helical reconstruction has many advantages over conventional Fourier-Bessel methods when dealing with variable helical symmetry and heterogeneity. We show here that when this method is applied to the ordered helical structure of the archebacterial uncoiled flagellar filament, significant extensions in resolution can be obtained readily when compared to applying traditional helical techniques. The filament population can be separated into classes of different morphologies, which may represent polymorphic states. Using cryo-negatively stained images, a resolution of approximately 10-15 A has been achieved. Single alpha-helices can be fit into the reconstruction, supporting the proposed similarity of the structure to that of type IV bacterial pili.