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We study computationally a family of β-hairpin peptides with systematically introduced chiral inversions, in explicit water, and we investigate the extent to which the backbone structure is able to fold in the presence of heterochiral perturbations. In contrast to the recently investigated case of a helical peptide, we do not find a monotonic change in secondary structure content as a function of the number of L- to D-inversions. The effects of L- to D-inversions are instead found to be highly position-specific. Additionally, in contrast to the helical peptide, some inversions increase the stability of the folded peptide: in such cases, we compute an increase in β-sheet content in the aqueous solution equilibrium ensemble. However, the tertiary structures of the stable (folded) configurations for peptides for which inversions cause an increase in β-sheet content show differences from one another, as well as from the native fold of the nonchirally perturbed β-hairpin. Our results suggest that although some chiral perturbations can increase folding stability, chirally perturbed proteins may still underperform functionally, given the relationship between structure and function.

Studying a set of helix-folding polyalanine peptides with systematically inserted chiral inversions in explicit water, we investigate quantitatively the effect of chiral perturbations on the structural ensembles of the peptides, thereby assessing the extent to which the backbone structure is able to fold in the presence of systematic heterochiral perturbations. Starting from the homochiral l-Ala20 peptide, we invert the backbone chiralities of Ala residues one by one along a specific perturbation pathway, until reaching the homochiral d-Ala20 peptide. Analysis of the helical contents of the simulated structural ensembles of the peptides shows that even a single inversion in the middle of the peptide completely breaks the helical structure in its vicinity and drastically reduces the helical content of the peptide. Further inversions in the middle of the peptide monotonically decrease the original helical content, that is, the right-handed helical content for l-Ala, and increase the helical content of the o...

Monomers of amyloid- β (A β ) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Forster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of A β isoforms with 40 (A β 40) and 42 residues (A β 42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ∼35 ns, similar to that of disordered proteins. These results suggest that both A β 40 and A β 42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies.

Formins accelerate actin polymerization, assumed to occur through flexible Formin Homology 1 (FH1) domain-mediated transfer of profilin-actin to the barbed end. To study FH1 properties and address sequence effects, including varying length/distribution of profilin-binding proline-rich motifs, we performed all-atom simulations of a set of representative FH1 domains of formins: mouse mDia1 and mDia2, budding yeast Bni1 and Bnr1, and fission yeast Cdc12, For3, and Fus1. We find FH1 has flexible regions between high-propensity polyproline helix regions. A coarse-grained model retaining sequence specificity, assuming rigid polyproline segments, describes their size. Multiple bound profilins or profilin-actin complexes expand mDia1-FH1, which may be important in cells. Simulations of the barbed end bound to Bni1-FH1-FH2 dimer show that the leading FH1 can better transfer profilin or profilin-actin, with decreasing probability as the distance from FH2 increases.

Formins accelerate actin polymerization, assumed to occur through flexible FH1 domain mediated transfer of profilin-actin to the barbed end. To study FH1 properties and address sequence effects including varying length/distribution of profilin-binding proline-rich motifs, we performed all-atom simulations of mouse mDia1, mDia2; budding yeast Bni1, Bnr1; fission yeast Cdc12, For3, and Fus1 FH1s. We find FH1 has flexible regions between high propensity polyproline helix regions. A coarse-grained model retaining sequence-specificity, assuming rigid polyproline segments, describes their size. Multiple profilins and profilin-actin complexes can simultaneously bind, expanding mDia1-FH1, which may be important in cells. Simulations of the barbed end bound to Bni1-FH1-FH2 dimer show the leading FH1 can better transfer profilin or profilin-actin, having decreasing probability with increasing distance from FH2.

Forster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely, the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble properties from FRET data, in which the polymer scaling exponent is allowed to vary with solution conditions. In its simplest form, it requires either the mean FRET efficiency or fluorescence lifetime information. In order to test the accuracy of the method, we have utilized both synthetic FRET data from implicit and explicit solvent simulations for 30 different protein sequences, and experimental single-molecule FRET data for an intrinsically disordered and a denatured protein. In all cases, we find that the inferred radii of gyration are within 10% of the true values, thus providing higher accuracy than simpler polymer models. In addition, the scaling exponents obtained by our procedure are in good agreement with those determined directly from the molecular ensemble. Our approach can in principle be generalized to treating other ensemble-averaged functions of intramolecular distances from experimental data.

We study the differences in folding stability of β-hairpin peptides, including GB1 hairpin and a point mutant GB1 K10G, as well as tryptophan zippers (TrpZips): TrpZip1, TrpZip2, TrpZip3-1, and TrpZip4. By performing replica-exchange molecular dynamics simulations with Amber03* force field (a modified version of Amber ff03) in explicit solvent, we observe ab initio folding of all the peptides except TrpZip3-1, which is experimentally known to be the least stable among the peptides studied here. By calculating the free energies of unfolding of the peptides at room temperature and folding midpoint temperatures for thermal unfolding of peptides, we find that TrpZip4 and GB1 K10G peptides are the most stable β-hairpins followed by TrpZip1, GB1, and TrpZip2 in the given order. Hence, the proposed K10G mutation of GB1 peptide results in enhanced stability compared to wild-type GB1. An important goal of our study is to test whether simulations with Amber 03* model can reproduce experimentally predicted folding stability differences between these peptides. While the stabilities of GB1 and TrpZip1 yield close agreement with experiment, TrpZip2 is found to be less stable than predicted by experiment. However, as heterogenous folding of TrpZip2 may yield divergent thermodynamic parameters by different spectroscopic methods, mismatching of results with previous experimental values are not conclusive of model shortcomings. For most of the cases, molecular simulations with Amber03* can successfully reproduce experimentally known differences between the mutated peptides, further highlighting the predictive capabilities of current state-of-the-art all-atom protein force fields.

Intrinsically disordered proteins (IDPs), which are expected to be largely unstructured under physiological conditions, make up a large fraction of eukaryotic proteins. Molecular dynamics simulations have been utilized to probe structural characteristics of these proteins, which are not always easily accessible to experiments. However, exploration of the conformational space by brute force molecular dynamics simulations is often limited by short time scales. Present literature provides a number of enhanced sampling methods to explore protein conformational space in molecular simulations more efficiently. In this work, we present a comparison of two enhanced sampling methods: temperature replica exchange molecular dynamics and bias exchange metadynamics. By investigating both the free energy landscape as a function of pertinent order parameters and the per-residue secondary structures of an IDP, namely, human islet amyloid polypeptide, we found that the two methods yield similar results as expected. We also highlight the practical difference between the two methods by describing the path that we followed to obtain both sets of data.

BACKGROUND The effects of designed methanol and co-substrate feeding strategies on fed-batch recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut+ were investigated. RESULTS The highest cell concentration was obtained as 157 g L−1 with the exponential feeding strategy where methanol was fed at a pre-calculated feeding rate based on pre-determined µ0 = 0.03 h−1, and mannitol concentration was kept constant at 50 g L−1 for t=0–6 h. The highest rhGH concentration was obtained as 1.3 g L−1 at t=54 h with the methanol-stat feeding strategy, where no co-substrate was used and the methanol concentration was kept at 5 g L−1 in the medium during the production phase. Thereafter, in another methanol-stat feeding strategy with pulse-fed sorbitol, the highest rhGH concentration was obtained as 1.2 g L−1 at t=42 h. CONCLUSION The methanol-stat fed-batch method keeping methanol concentration constant at 5 g L−1 in the production medium increased rhGH production at least 2-fold compared with that of the exponential feeding strategies. In the designed methanol-stat feeding strategies, use of sorbitol as co-substrate decreased methanol demand of the cells 2-fold, increased the overall product yield on the substrate 1.6-fold, shortened the cultivation time by 12 h and increased the productivity. Thus, methanol-stat feeding with co-substrate sorbitol was found to be a favorable fed-batch strategy for rhGH production by Pichia pastoris under alcohol oxidase I promoter. © 2014 Society of Chemical Industry

Amyloid aggregates are characteristic of many serious diseases such as Alzheimer's disease, Parkinson's, and type 2 diabetes and commonly involve intrinsically disordered proteins (IDPs), those that populate an ensemble of conformations rather than a single folded structure. Human islet amyloid polypeptide (hIAPP or amylin) is an amyloidogenic IDP implicated in pancreatic β-cell death during the pathogenesis of type 2 diabetes. The target of amylin's toxic activity is thought to be the cell's lipid membrane, which may also act as a catalyst for aggregation. Since amylin is intrinsically disordered, differing environments can have a large impact on its equilibrium conformational ensemble. We apply atomistic molecular dynamics simulations on multiple systems containing a full-length amylin monomer and a lipid bilayer to study the changes induced by the membrane. We observe stabilized helical conformations structurally similar to those determined by NMR experiments conducted in similar environments. We also find that bilayers of different compositions result in greatly different equilibrium ensembles of amylin. Finally, we discuss how a mixed bilayer containing zwitterionic and anionic lipid headgroups can allow for greater preference toward conformations which are adsorbed below the membrane surface through rearrangement of lipids for more favorable protein-lipid interactions.

Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ2, has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances.

Human islet amyloid polypeptide (hIAPP), a 37-residue protein cosecreted with insulin by β-cells in the pancreas, is known to form amyloid fibrils in type II diabetes patients. This fibril formation is also associated with β-cell death. However, rat IAPP (rIAPP) does not aggregate into fibrils, nor is it associated with β-cell toxicity. Determining solution properties of hIAPP experimentally is difficult because it aggregates quickly. Our study uses molecular dynamics simulation to explore and compare in-solution characteristics of hIAPP and rIAPP, as well as two single-point hIAPP mutants, hIAPP I26P and hIAPP S20G, which exhibit observed differences from hIAPP in aggregation propensities. We find that all four polypeptide monomers sample structured states in solution. More importantly, differences in the helicity over residues 7-16 may play an important role in early aggregation, although this region is outside of commonly assumed amyloidogenic region 20-29. The long-range contacts, though unexpected of IDPs, cause variation in sampled conformations among four polypeptides within same amino acid sequence. Our results also yield evidence that previously determined structures bound to micelles are also transiently sampled in the solution state. In particular, similarities found in region 8-17 together with the helical differences that we observe in region 7-16 lead us to suggest that the region 7-16 is potentially responsible for amyloidogenic behavior of amylin peptides. Our results also provide support for the proposed mechanism of fibril formation based on experimentally observed transient helices in amyloidogenic peptides.

BACKGROUND Effects of co-substrate sorbitol different feeding strategies on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut+ were investigated by eight designed experiments grouped as: (i) fed-batch methanol feeding without the co-substrate; (ii) fed-batch methanol feeding with pulse sorbitol feeding; (iii) fed-batch methanol feeding together with fed-batch sorbitol feeding at t = 0–15 h, followed by fed-batch methanol feeding; and (iv) fed-batch methanol and sorbitol feeding at t = 0–30 h, followed with fed-batch methanol feeding. RESULTS The highest rhGH and cell concentrations were achieved, respectively, as 0.64 g L−1 and 105 g L−1 at t = 42 h of induction phase, with the strategy where methanol was fed to the system at a pre-determined feeding rate of μM0=0.03 h−1, and sorbitol concentration was kept at 50 g L−1 at t = 0–15 h of the rhGH production phase where the specific growth rate on sorbitol was μS0=0.025 h−1. The overall cell and product yield on total substrate were found as 0.26 g g−1 and 2.26 mg g−1, respectively. CONCLUSION This work demonstrates that co-carbon source, sorbitol, feeding strategy is as important as methanol feeding strategy in recombinant protein production by Mut+ strains of P. pastoris. © 2012 Society of Chemical Industry

Translating sticky biological molecules-such as mussel foot proteins (MFPs)-into synthetic, cost-effective underwater adhesives with adjustable nano- and macroscale characteristics requires an intimate understanding of the glue's molecular interactions. To help facilitate the next generation of aqueous adhesives, we performed a combination of surface forces apparatus (SFA) measurements and replica-exchange molecular dynamics (REMD) simulations on a synthetic, easy to prepare, Dopa-containing peptide (MFP-3s peptide), which adheres to organic surfaces just as effectively as its wild-type protein analog. Experiments and simulations both show significant differences in peptide adsorption on CH3-terminated (hydrophobic) and OH-terminated (hydrophilic) self-assembled monolayers (SAMs), where adsorption is strongest on hydrophobic SAMs because of orientationally specific interactions with Dopa. Additional umbrella-sampling simulations yield free-energy profiles that quantitatively agree with SFA measurements and are used to extract the adhesive properties of individual amino acids within the context of MFP-3s peptide adhesion, revealing a delicate balance between van der Waals, hydrophobic, and electrostatic forces.

Calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) are two intrinsically disordered proteins (IDP) of the calcitonin peptide family. CGRP is an important new target for antimigraine drugs, it is involved in vasodilation and transmission of pain signals. IAPP regulates blood glucose levels and forms amyloid fibers in type II diabetes. They share up to 40% sequence similarity and bind to very similar receptors, but are found in different types of tissue and carry out different functions. Recent experimental studies measuring end-to-end contact formation by tryptophan triplet quenching, show a significant difference in the conformational dynamics of CGRP and IAPP. We investigate this through Dynamic Flexibility Index (DFI). DFI quantifies the resilience of a given position to the perturbations that occur at different parts of the chain, using linear response theory. This captures multi-dimensional effects when the chain is displaced out of equilibrium. Thus, it identifies flexible and rigid positions that govern the function. Our DFI profiles obtained from equilibrated atomistic MD simulations reveal distinct patterns for CGRP and IAPP. We investigate the effect of amino acid substitutions on the conformational dynamics of the unbound disordered IDP, and pinpoint the positions that give a significant difference in dynamics.

hnRNPA2, a component of RNA-processing membraneless organelles, forms inclusions when mutated in a syndrome characterized by the degeneration of neurons (bearing features of amyotrophic lateral sclerosis [ALS] and frontotemporal dementia), muscle, and bone. Here we provide a unified structural view of hnRNPA2 self-assembly, aggregation, and interaction and the distinct effects of small chemical changes-disease mutations and arginine methylation-on these assemblies. The hnRNPA2 low-complexity (LC) domain is compact and intrinsically disordered as a monomer, retaining predominant disorder in a liquid-liquid phase-separated form. Disease mutations D290V and P298L induce aggregation by enhancing and extending, respectively, the aggregation-prone region. Co-aggregating in disease inclusions, hnRNPA2 LC directly interacts with and induces phase separation of TDP-43. Conversely, arginine methylation reduces hnRNPA2 phase separation, disrupting arginine-mediated contacts. These results highlight the mechanistic role of specific LC domain interactions and modifications conserved across many hnRNP family members but altered by aggregation-causing pathological mutations.

Glycosylation is one of the most common post-translational modifications (PTMs), which provides a large proteome diversity. Previous work on glycosylation of globular proteins has revealed remarkable effects of glycosylation on protein function, altering the folding stability and structure and/or altering the protein surface which affects their binding characteristics. Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of large proteins are also frequently glycosylated, yet how glycosylation affects their function remains to be elucidated. An important open question is, does glycosylation affect IDP structure or binding characteristics or both? In this work, we particularly address the structural effects of O-linked glycosylation by investigating glycosylated and unglycosylated forms of two different IDPs, tau174-183 and human islet amyloid polypeptide (hIAPP), by all-atom explicit solvent simulations. We simulate these IDPs in aqueous solution for O-linked glycosylated and unglycosylated forms by employing two modern all-atom force fields for which glycan parameters are also available. We find that O-linked glycosylation only has a modest effect on equilibrium structural ensembles of IDPs, for the cases studied here, which suggests that the functional role of glycosylation may be primarily exerted by modulation of the protein binding characteristics rather than structure.

We use all-atom molecular simulation with explicit solvent to study the properties of selected intrinsically disordered proteins and unfolded states of foldable proteins, which include chain dimensions and shape, secondary structure propensity, solvent accessible surface area, and contact formation. We find that the qualitative scaling behavior of the chains matches expectations from theory under ambient conditions. In particular, unfolded globular proteins tend to be more collapsed under the same conditions than charged disordered sequences of the same length. However, inclusion of explicit solvent in addition naturally captures temperature-dependent solvation effects, which results in an initial collapse of the chains as temperature is increased, in qualitative agreement with experiment. There is a universal origin to the collapse, revealed in the change of hydration of individual residues as a function of temperature: namely, that the initial collapse is driven by unfavorable solvation free energy of individual residues, which in turn has a strong temperature dependence. We also observe that in unfolded globular proteins, increased temperature also initially favors formation of native-like (rather than non-native-like) structure. Our results help to establish how sequence encodes the degree of intrinsic disorder or order as well as its response to changes in environmental conditions.

Experiments measuring contact formation between a probe and quencher in disordered chains provide information on the fundamental dynamical timescales relevant to protein folding, but their interpretation usually relies on simplified one-dimensional (1D) diffusion models. Here, we use all-atom molecular simulations to capture both the time-scales of contact formation, as well as the scaling with the length of the peptide for tryptophan triplet quenching experiments. Capturing the experimental quenching times depends on the water viscosity, but more importantly on the configurational space explored by the chain. We also show that very similar results are obtained from Szabo-Schulten-Schulten theory applied to a 1D diffusion model derived from the simulations, supporting the validity of such models. However, we also find a significant reduction in diffusivity at small separations, those which are most important in determining the quenching rate., 12 pages, 5 figures

We study the differences in folding stability of β-hairpin peptides, including GB1 hairpin and a point mutant GB1 K10G, as well as tryptophan zippers (TrpZips): TrpZip1, TrpZip2, TrpZip3-1, and TrpZip4. By performing replica-exchange molecular dynamics simulations with Amber03* force field (a modified version of Amber ff03) in explicit solvent, we observe ab initio folding of all the peptides except TrpZip3-1, which is experimentally known to be the least stable among the peptides studied here. By calculating the free energies of unfolding of the peptides at room temperature and folding midpoint temperatures for thermal unfolding of peptides, we find that TrpZip4 and GB1 K10G peptides are the most stable β-hairpins followed by TrpZip1, GB1, and TrpZip2 in the given order. Hence, the proposed K10G mutation of GB1 peptide results in enhanced stability compared to wild-type GB1. An important goal of our study is to test whether simulations with Amber 03* model can reproduce experimentally predicted folding stability differences between these peptides. While the stabilities of GB1 and TrpZip1 yield close agreement with experiment, TrpZip2 is found to be less stable than predicted by experiment. However, as heterogenous folding of TrpZip2 may yield divergent thermodynamic parameters by different spectroscopic methods, mismatching of results with previous experimental values are not conclusive of model shortcomings. For most of the cases, molecular simulations with Amber03* can successfully reproduce experimentally known differences between the mutated peptides, further highlighting the predictive capabilities of current state-of-the-art all-atom protein force fields.

The yeast Pichia (Komagataella) pastoris has become a potential host system for recombinant protein (r-protein) production. Several strong inducible and constitutive promoters have been identified in P. pastoris which make the host ideal in r-protein expressions. The constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene. P-GAp, is promising as its use offers the advantage of associating product quantity to growth which can be optimized by a well-designed feeding strategy so long as the product itself is not toxic to the host. In this article, carbon source metabolism and the central carbon pathways of P. pastoris are reviewed. After comparing P-GAp with the available promoters of P. pastoris, the bioreactor operation parameters and bioreactor operation strategies are analyzed for r-protein production under P-GAp by focusing on response of the cells, final product concentration, yield, and productivity. The overview of the subject reveals the requirement for in-depth information on the overall regulation of P. pastoris metabolism that can enable fine-tuning bioreactor operation parameters in relation with the physiology of the microorganism in order to develop new strategies for the enhanced r-protein production. (C) 2014 Elsevier B.V. All rights reserved.

MicroRNAs and other small oligonucleotides in biofluids are promising biomarkers, but conventional assays require complex processing steps unsuitable for point-of-care assays or implantable/wearable sensors. Single-walled carbon nanotubes are an ideal material for implantable sensors due to emission in the near-infrared spectral region, photostability, and exquisite sensitivity. We engineered a carbon nanotube-based platform capable of real-time optical quantification of hybridization events of microRNA and other oligonucleotides for use in vivo. The sensor mechanism derived from competitive effects between displacement of both oligonucleotide charge groups and water from the nanotube surface, resulting in a solvatochromism-like response. The platform allowed detection via single molecule sensor elements and multiplexing using multiple nanotube species. The sensor monitored toehold-based strand displacement events, reversing the sensor response and regenerating the sensor complex. The sensor functioned in whole urine and serum, and it non-invasively measured DNA and microRNA biomarkers after implantation into live mice. Citation Format: Daniel A. Heller, Jackson D. Harvey, Prakrit V. Jena, Ryan M. Williams, Thomas V. Galassi, Hanan A. Baker, Daniel Roxbury, Gül Zerze, Jeetain Mittal. A nanoscale optical reporter implant for miRNA biomarkers in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-222. doi:10.1158/1538-7445.AM2017-LB-222

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Concentration and composition of ciliate protozoa in the families Ophryoscolecidae and Isotrichidae were determined in rumen contents of domestic sheep (Ovis ammon aries) from Cyprus. A total of five genera of Ophryoscolecidae were identified, Metadinium, Enoploplastron, Polyplastron, Epidinium, and Ophrvoscolex, which included six species: Metadinium affine, Enoploplastron triloricatum, Polyplastron multivesiculatum, Epidinium ecaudatum, Epidinium graini, and Ophryoscolex purkynjei. Eight separate forms of Epidinium were identified (E. ecaudatum f. ecaudatum, E. e. f. caudatum, E. e. f. bicaudatum, E. e. f. tricaudatum, E. e. f. quadricaudatum, E. graini f. graini, E. g. f. caudatricoronatum, and E. g. f. caudaquadricoroniatum), along with five forms of Ophryoscolex purkynjei (O. p. f. purkynjei, O. p. f. bifidobicinctus, O. p. f. bifidoquadricinctus, O. p. f. bicoronatus, O. p. f. tricoronatus, and O. p. f. quadricoronatus). Three species of Isotrichidae were observed, Isotricha intestinalis, I. prostoma, and Dasytricha ruminantium. This study reports new host records for three forms of Epidinium graini and Ophryoscolex purkynjei f. bifidobicinctus. The rumen fauna in the family Ophryoscolecidae from Cypriote domestic sheep appear to have limited diversity compared to those from Turkish and Far Eastern (Chinese/Japanese) sheep, while they are more diverse than those found in Western European (Scottish) and North American (Canadian/Alaskan) sheep.