Modeling Adsorption, Conformation, and Orientation of the Fis1 Tail Anchor at the Mitochondrial Outer Membrane.

Proteins can be targeted to organellar membranes by using a tail anchor (TA), a stretch of hydrophobic amino acids found at the polypeptide carboxyl-terminus. The Fis1 protein (Fis1p), which promotes mitochondrial and peroxisomal division in the yeast Saccharomyces cerevisiae, is targeted to those organelles by its TA. Substantial evidence suggests that Fis1p insertion into the mitochondrial outer membrane can occur without the need for a translocation machinery. However, recent findings raise the possibility that Fis1p insertion into mitochondria might be promoted by a proteinaceous complex. Here, we have performed atomistic and coarse-grained molecular dynamics simulations to analyze the adsorption, conformation, and orientation of the Fis1(TA). Our results support stable insertion at the mitochondrial outer membrane in a monotopic, rather than a bitopic (transmembrane), configuration. Once inserted in the monotopic orientation, unassisted transition to the bitopic orientation is expected to be blocked by the highly charged nature of the TA carboxyl-terminus and by the Fis1p cytosolic domain. Our results are consistent with a model in which Fis1p does not require a translocation machinery for insertion at mitochondria.

Structure-based engineering of an antiangiogenic scFv antibody for soluble production in E. coli without loss of activity.

Development of monoclonal antibody therapeutics against vascular endothelial growth factor receptor 2 (VEGFR-2) protein, which is the main regulator in angiogenesis, has been a major challenge for years. In the current study, we engineer an inclusion body forming single-chain variable fragment (scFv) against VEGFR-2 by using complementarity determining regions (CDR) grafting technique to improve its solubility and investigate the activity of the engineered molecule. CDR sequences of the target scFv were grafted into the framework of another intrinsically soluble scFv molecule. Based on the computational results, CDR grafting has increased the solubility of the grafted scFv molecule. Results confirmed that the grafting approach increased in vivo folding properties of the target scFv molecule compared with the original scFv molecule. Similar binding affinities to the VEGFR-2 were observed for the original and the grafted scFv by surface plasmon resonance assays. Biological activity assays, including human umbilical vein endothelial cells proliferation and wound healing assays, showed that grafted scFv molecule has an antiangiogenic property. This study suggests that an antiangiogenic scFv fully expressed as an inclusion body can be rescued by grafting its CDR regions to a scFv expressed in a soluble form without any loss in its binding property and its activity.

Representation of the conformational ensemble of peptides in coarse grained simulations.

In their native state, many proteins/peptides display an ensemble of conformations, rather than a unique tertiary structure. Novel experimental techniques have enabled a quantitative analysis of this structural heterogeneity. In molecular dynamics simulations, however, capturing this conformational ensemble quantitatively remains a major challenge even with all atom simulations. In coarse grained (CG) simulations, with fewer degrees of freedom, representation of the conformational ensemble becomes more problematic. Here, we revisit a CG model from our group, which was designed to address the conformational transferability problem by using the LKα14 peptide as a model system. The LKα14 peptide transitions from a random/unstructured state in dilute solution to a solely α-helical conformation upon aggregation as evidenced by circular dichroism. Here, we demonstrate that the structure/physics based approach, used in the original parameterization of our CG model, strongly depends on the reference system chosen and excluded volume interactions that are often considered to be of secondary importance. We first tune the excluded volume parameters by using both α-helix and ß-sheet type structures as reference and then update the nonbonded interactions by using a goodness-of-fit metric for representation of the conformational ensemble of LKα14. We demonstrate that the updated model can recover the whole conformational ensemble quantitatively while maintaining the aggregation driven conformational transition. This balanced parametrization with regard to alternative secondary structures opens the door for the generalization of the CG model to other sequences, which we demonstrate on a ß-sheet forming triblock peptide.

Assembly of Huntingtin headpiece into α-helical bundles.

Protein aggregation is a hallmark of neurodegenerative disorders. In this group of brain-related disorders, a disease-specific "host" protein or fragment misfolds and adopts a metastatic, aggregate-prone conformation. Often, this misfolded conformation is structurally and thermodynamically different from its native state. Intermolecular contacts, which arise in this non-native state, promote aggregation. In this regard, understanding the molecular principles and mechanisms that lead to the formation of such a non-native state and further promote the formation of the critical nucleus for fiber growth is essential. In this study, the authors analyze the aggregation propensity of Huntingtin headpiece (httNT), which is known to facilitate the polyQ aggregation, in relation to the helix mediated aggregation mechanism proposed by the Wetzel group. The authors demonstrate that even though httNT displays a degenerate conformational spectrum on its own, interfaces of macroscopic or molecular origin can promote the α-helix conformation, eliminating all other alternatives in the conformational phase space. Our findings indicate that httNT molecules do not have a strong orientational preference for parallel or antiparallel orientation of the helices within the aggregate. However, a parallel packed bundle of helices would support the idea of increased polyglutamine concentration, to pave the way for cross-ß structures.

Role of Hydrophobic/Aromatic Residues on the Stability of Double-Wall ß-Sheet Structures Formed by a Triblock Peptide.

Bioinspired self-assembling peptides serve as powerful building blocks in the manufacturing of nanomaterials with tailored features. Because of their ease of synthesis, biocompatibility, and tunable activity, this emerging branch of biomolecules has become very popular. The triblock peptide architecture designed by the Hartgerink group is a versatile system that allows control over its assembly and has been shown to demonstrate tunable bioactivity. Three main forces, Coulomb repulsion, hydrogen bonding and hydrophobicity act together to guide the triblock peptides' assembly into one-dimensional objects and hydrogels. It was shown previously that both the nanofiber morphology (e.g., intersheet spacing, formation of antiparallel/parallel ß-sheets) and hydrogel rheology strictly depend on the choice of the core residue where the triblock peptide fibers with aromatic cores in general form shorter fibers and yield poor hydrogels with respect to the ones with aliphatic cores. However, an elaborate understanding of the molecular reasons behind these changes remained unclear. In this study, by using carefully designed computer based free energy calculations, we analyzed the influence of the core residue on the formation of double-wall fibers and single-wall ß-sheets. Our results demonstrate that the aromatic substitution impairs the fiber cores and this impairment is mainly associated with a reduced hydrophobic character of the aromatic side chains. Such weakening is most obvious in tryptophan containing peptides where the fiber core absorbs a significant amount of water. We also show that the ability of tyrosine to form side chain hydrogen bonds plays an indispensable role in the fiber stability. As opposed to the impairment of the fiber cores, single-wall ß-sheets with aromatic faces become more stable compared to the ones with aliphatic faces suggesting that the choice of the core residue can also affect the underlying assembly mechanism. We also provide an in-depth comparison of competing structures (zero-dimensional aggregates, short and long fibers) in the triblock peptides' assembly and show that by adjusting the length of the terminal blocks, the fiber growth can be turned on or off while keeping the nanofiber morphology intact.

Relation between Protein Intrinsic Normal Mode Weights and Pre-Existing Conformer Populations.

Intrinsic fluctuations of a protein enable it to sample a large repertoire of conformers including the open and closed forms. These distinct forms of the protein called conformational substates pre-exist together in equilibrium as an ensemble independent from its ligands. The role of ligand might be simply to alter the equilibrium toward the most appropriate form for binding. Normal mode analysis is proved to be useful in identifying the directions of conformational changes between substates. In this study, we demonstrate that the ratios of normalized weights of a few normal modes driving the protein between its substates can give insights about the ratios of kinetic conversion rates of the substates, although a direct relation between the eigenvalues and kinetic conversion rates or populations of each substate could not be observed. The correlation between the normalized mode weight ratios and the kinetic rate ratios is around 83% on a set of 11 non-enzyme proteins and around 59% on a set of 17 enzymes. The results are suggestive that mode motions carry intrinsic relations with thermodynamics and kinetics of the proteins.

Assembly of Triblock Amphiphilic Peptides into One-Dimensional Aggregates and Network Formation.

Peptide assembly plays a key role in both neurological diseases and development of novel biomaterials with well-defined nanostructures. Synthetic model peptides provide a unique platform to explore the role of intermolecular interactions in the assembly process. A triblock peptide architecture designed by the Hartgerink group is a versatile system which relies on Coulomb interactions, hydrogen bonding, and hydrophobicity to guide these peptides' assembly at three different length scales: ß-sheets, double-wall ribbon-like aggregates, and finally a highly porous network structure which can support gels with ≤1% by weight peptide concentration. In this study, by using molecular dynamics simulations of a structure based implicit solvent coarse grained model, we analyzed this hierarchical assembly process. Parametrization of our CG model is based on multiple-state points from atomistic simulations, which enables this model to represent the conformational adaptability of the triblock peptide molecule based on the surrounding medium. Our results indicate that emergence of the double-wall ß-sheet packing mechanism, proposed in light of the experimental evidence, strongly depends on the subtle balance of the intermolecular forces. We demonstrate that, even though backbone hydrogen bonding dominates the early nucleation stages, depending on the strength of the hydrophobic and Coulomb forces, alternative structures such as zero-dimensional aggregates with two ß-sheets oriented orthogonally (which we refer to as a cross-packed structure) and ß-sheets with misoriented hydrophobic side chains are also feasible. We discuss the implications of these competing structures for the three different length scales of assembly by systematically investigating the influence of density, counterion valency, and hydrophobicity.

Self-assembling multidomain peptide fibers with aromatic cores.

Self-assembling multidomain peptides have been shown to have desirable properties, such as the ability to form hydrogels that rapidly recover following shear-thinning and the potential to be tailored by amino acid selection to vary their elasticity and encapsulate and deliver proteins and cells. Here we describe the effects of substitution of aliphatic hydrophobic amino acids in the central domain of the peptide for the aromatic amino acids phenylalanine, tyrosine, and tryptophan. While the basic nanofibrous morphology is retained in all cases, selection of the particular core residues results in switching from antiparallel hydrogen bonding to parallel hydrogen bonding in addition to changes in nanofiber morphology and in hydrogel rheological properties. Peptide nanofiber assemblies are investigated by circular dichroism polarimetry, infrared spectroscopy, atomic force microscopy, transmission and scanning electron microscopy, oscillatory rheology, and molecular dynamics simulations. Results from this study will aid in designing next generation cell scaffolding materials.