Structural analysis of cross α-helical nanotubes provides insight into the designability of filamentous peptide nanomaterials

The exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili

Synthetic biology approaches to direct recombinant protein filament assembly

The design of self-assembling modular nanoscale devices from biological materials has long been a goal of synthetic biology. Filamentous protein assemblies have been investigated as modular scaffolds that can be functionalised through genetic or chemical fusion of the protein subunits to other molecules. The bioengineer’s toolbox is replete with diverse natural protein filaments, with each being suited to applications for different functions and at different scales. However, the length distribution inherent within filamentous protein assemblies has precluded their application for precise nanoscale designs. Efforts to control the length of protein filaments have included the application of capping proteins, which prevent further elongation when incorporated into an assembling filament. To extend and perfect this strategy, immobilising capping proteins within a defined cavity, where they would serve as nucleation sites for filament assembly, would enable precise control over the length of the resulting filament. To create such a confined space, the predictable nature of Watson-Crick base pairing could be harnessed to design and produce nanoscale moulds using DNA origami. Within this thesis, we addressed several challenges in protein and DNA engineering that must be overcome to achieve this vision. In the first results chapter, an existing capping protein was converted into a split-protein system with the vision of directing protein filament nucleation. In the second results chapter, novel capping proteins for two thermostable protein filaments were designed, produced, and tested. In the third results chapter, a nanoscale mould was constructed using DNA origami. Together, the results presented in this thesis suggest that guiding protein filament assembly with nucleation proteins is a plausible means of controlling filament length

Cell-Penetrating Peptides: design strategies beyond primary structure and amphipathicity

Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies

Engineering of coiled-coil protein scaffolds as innovative tools for biosensing applications

2007/2008A new generation of protein scaffolds is becoming a valid alternative tool to recombinant antibodies of biotechnological, medical and pharmaceutical applications, where strong affinity and specificity are required. They share with antibodies important features (target affinity and specificity), but they have also some improvements (smaller size of molecule, tolerance to modification of the framework and the recognition site restricted to few residues), that can be exploited for biosensing application in nanotechnological platforms. Nanotechnology has been played an increasingly important role in the development of biosensors, improving the intrinsic features of biodevices. In this thesis work, we analyzed the coiled-coil domain, a widely spread dimerization domain shared by several protein scaffolds, and involved in protein-protein interaction in both eukaryotic and prokaryotic cells. The analysis of the coiled-coil structure allows a de novo design of new peptides, namely E and K, that can dimerize as a E/K coiled-coil system: the dimerization feature and the stability of the interaction makes this system an ideal platform to build up functional and customizable biosensors. A characterization of the E/K interaction was performed by using the protein complementation assay (PCA), a useful biological method to investigate the interaction between protein partners. With this in vivo method, we corroborate the interaction features determinate with circular dichroism, and we demonstrated that E and K coils effectively represent a protein scaffold, able to tolerate amino acid substitutions without altering its main structure. In addition, we create two libraries of K mutant coils, randomizing the peptide sequence, and with PCA we selected new K binders (Kran 5.17 and Krd F8) that showed a comparable interaction activity with the E-coil in preliminary in vitro tests. In the last part of this work, we generate a library of a new scaffold molecule (the single chain E-K) capable to bind small molecules as a single protein product containing both domains. Using the phage display selection system, we isolated scsE-K that can bind our analyte (the caffeine) with high specificity. This new molecules can be a powerful tool for analytical and biomedical applications.XXI Ciclo198

A study of M0R1/GEM1 and kinl-like kinesins arabidopsis

Microtubules perform essential functions in eukaryotic cells and, with other cytoskeletal elements, are involved in diverse cellular processes. Plant cells construct four microtubule arrays; There are two cortical arrays, the interphase cortical array, and the preprophase band, the mitotic spindle and the cytokinetic phragmoplast. The control and rearrangement of these arrays through the cell cycle is coordinated by Microtubule Associated Proteins or MAPs. These proteins have diverse functions including anchoring & crosslinking microtubules or otherwise regulating microtubule formation and destruction within the cell. MOR1/GEM1 is the Arabidopsis member of the conserved XMAP215/TOGp family of proteins which are microtubule stablising MAPs and promote microtubule polymerization in vitro. In many organisms such stabilization is opposed by Kinl catastrophic kinesins, which depolymerise and destabilize microtubules. In addition to the further characterization of MOR1/GEM1, here in this thesis, two putative KinI kinesins are identified in the Arabidopsis genome, a subfamily of kinesins previously uncharacterized in plants, AtCMK1 & AtCMK2. Immunolocalisation shows that MOR1/GEM1 associates with all plant microtubule arrays throughout the cell cycle and Is found to concentrate at the plus end of microtubules In the spindle next to chromosomes and the midline of the phragmoplast where oppositely orientated microtubules overlap. Furthermore, consistent with this localization, we show that a C-terminal fragment of MOR1/GEM1 which Is absent in the pollen cytokinesis mutant, gem1 can bind microtubules and as such we propose that the defects in the phragmoplast In gem1 mutant is a result of the reduction of the MOR1/GEM1 protein to bind microtubules. Further studies indicate that unlike its budding yeast homologue Stu2, MOR1/GEM1 does not for dimers.Immunolocalisation shows that AtCMK2 associates with all the plant microtubule arrays throughout the cell cycle, particularly the metaphase spindle. However in our experiments, AtCMKI does not, but rather locates to structures within the cytoplasm, such as golgi vesicles or organelles. Here we also show that AtCMK kinesins form homodimers and do not physically Interact with MOR1/GEM1 suggesting that these factors may be genetic interactors instead. Over-expression of AtCMK2 as a GPP fusion protein results in the disruption of microtubules, leaving short microtubule fragments and tubulin oligomers or aggregates, suggesting that AtCMK2 Is a true Arabidopsis catastrophic kinesin. In addition, GUS promoter fusions show that the expression patterns of these two kinesins are very different

Crystal structure determination of HEX1 and human GEMININ 70-152

Ph.DDOCTOR OF PHILOSOPH

Biocatalysts based on peptide and peptide conjugate nanostructures

Peptides and their conjugates (to lipids, bulky N-terminals, or other groups) can self-assemble into nanostructures such as fibrils, nanotubes, coiled coil bundles, and micelles, and these can be used as platforms to present functional residues in order to catalyze a diversity of reactions. Peptide structures can be used to template catalytic sites inspired by those present in natural enzymes as well as simpler constructs using individual catalytic amino acids, especially proline and histidine. The literature on the use of peptide (and peptide conjugate) α-helical and β-sheet structures as well as turn or disordered peptides in the biocatalysis of a range of organic reactions including hydrolysis and a variety of coupling reactions (e.g., aldol reactions) is reviewed. The simpler design rules for peptide structures compared to those of folded proteins permit ready ab initio design (minimalist approach) of effective catalytic structures that mimic the binding pockets of natural enzymes or which simply present catalytic motifs at high density on nanostructure scaffolds. Research on these topics is summarized, along with a discussion of metal nanoparticle catalysts templated by peptide nanostructures, especially fibrils. Research showing the high activities of different classes of peptides in catalyzing many reactions is highlighted. Advances in peptide design and synthesis methods mean they hold great potential for future developments of effective bioinspired and biocompatible catalysts

Characterization of Desmin Disease Mutants and their Association with alphaB-Crystallin in Desminopathy

Mutations in intermediate filaments (IFs) and associated proteins have been shown to cause a number of diseases in humans, ranging from blistering skin diseases to premature aging, as well as from cataract to cardiomyopathies. Desminopathy, a disease caused by dysfunctional mutations in type III muscle-specific IF protein desmin, constitutes a distinct sub-group of myofibrillar myopathies (MFM) manifesting as skeletal and / or cardiac myopathy. Mutations in the chaperone aB-crystallin, that supposedly maintains cytoskeletal integrity, have also been identified to cause MFM. Intracytoplasmic aggregates in desminopathy uniformly comprise aberrantly folded desmin that, among other proteins, recruits aB-crystallin. Currently, the molecular basis of sequential events that lead to such aggregates in the myocyte of patients harboring desmin mutations is not well understood. Thus, to unravel the molecular basis of desminopathy, we have investigated the interdependence between filament alterations arising from desmin mutations and their functional consequences in terms of interaction with the small heat shock protein aB-crystallin. We have systematically characterized various mutants spanning the non-a-helical aminoterminal “head”, central a-helical “rod” and non-a-helical carboxy-terminal “tail” domain of desmin. We show by in vitro characterization of the five head mutants, that two mutant variants residing in the conserved nonapeptide motif “SSYRRTFGG” of desmin - DesS13F and DesR16C - interfere with assembly by forming filamentous aggregates. Consistent with in vitro data, both mutants fail to generate a bona fide filament system in cells lacking a type III IF cytoskeleton. In cells expressing vimentin or desmin, both mutants fail to integrate into the endogenous filament network and severely affect its cellular localization. The novel desminopathy-causing mutant DesL377del22fs apparently interacts with wild-type desmin at dimer, tetramer and higher level of filament organization in vitro, but leads to a disruption of the IF cytoskeleton in cells. This mutant is not detectable in the myotubes of a heterozygous carrier even upon proteasome inhibition. Two – DesR454W and DesK449T - out of the six tail mutants form abnormal filaments during in vitro assembly and correspondingly generate aberrant filaments in cells devoid of type III IF protein cytoskeleton. The desmin fragment Des(ESA)delC244, resembling almost “first-half” of a desmin molecule, has deleterious effects on filament assembly in vitro as well as in transfected cells. It exhibits nucleoplasmic aggregates in two of the four investigated cell lines. With respect to characterizing the association of desmin disease mutants with aB-crystallin, data from yeast two-hybrid analyses, electron microscopy (EM), cosedimentation assay and viscometry distinctly suggest that the tail domain of desmin is pivotal in modulating its binding to aB-crystallin. We show that aB-crystallin binds to wild-type desmin filament under optimized buffer condition, but its binding to C-terminal deletion variants is either diminished or abolished. We speculate that this occurs due to differences in hydrophobic surface properties and exposed residues of wild-type desmin and its deletion variants. DesdelRDG is devoid of the conserved tripeptide motif “RDG”, yet it binds to aB-crystallin with similar strength as desmin wild-type. Thus, we propose that the prerequisite for binding of aB-crystallin to desmin is the 3-dimensional desmin protein conformation, which can be altered due to a mutation, and not the linear amino acid sequence involving conserved motifs per se. The two tail mutants – DesI451M and DesR454W - reveal weaker and stronger binding, respectively, to aB-crystallin as compared to wild-type protein. With respect to kinetics of binding, unlike desmin wild-type, DesR454W binds to aB-crystallin at all stages of assembly, and this probably results from its “open” filament structure both alone and in an equimolar mixture with wild-type desmin. Data from R454W and wild-type desmin transfection in 3T3 cells corroborate the in vitro data, showing that DesR454W binds around 50% more cytosolic aB-crystallin than desmin wild-type. Hence, our data suggest that mutations in desmin cause toxic gain-of-function, whereby the desmin mutants show enhanced binding to aB-crystallin. A plausible explanation for aggregate formation in desminopathy could be such modified protein-protein interactions. In summary, our data demonstrate the impact of desmin mutations not only on its structural property, but also on its molecular interaction with aB-crystallin. This adds to our understanding of the molecular basis of desminopathy as we show for the first time that subtle alterations in the nanoarchitecture of desmin filament are sufficient to induce aberrant interaction with an associated protein aB-crystallin. Such a modification might eventually contribute to the pathogenesis of desminopathy

Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: A Dissertation

Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function

Structural investigation of the molecular mechanisms underlying titin elasticity and signaling

Titin is a giant protein that spans >1µm from the Z-disc to the M-line, forming an intrasarcomeric filament system in vertebrate striated muscle, which is not only essential for the assembly of the sarcomere, but also critical for myofibril signaling and metabolism. Furthermore, it provides the sarcomere with resting tension, elasticity and restoring forces upon stretch, ensuring the correct positioning of the actin-myosin motors during muscle function. Titin is composed of ~300 immunoglobulin (Ig) and fibronectin-III (FnIII) domains, arranged in linear tandems. They are interspersed by an auto-inhibited Ser kinase (TK) close to its C-terminus as well as several unique sequences, most prominently a differentially spliced stretch rich in PEVK residues which localizes to the I-band part of titin where its elastic properties reside. There, the PEVK segment is flanked by a long Ig tandem, which together act as serial molecular springs that determine titin elastic response. The focus of this work lay in the elucidation of the molecular mechanisms governing titin I-band elasticity and the recruitment of the M-line signalosome around TK involved in the control of myofibril turnover and the trophic state of muscle. To that effect, we have elucidated the crystal structure of a six-Ig fragment representative of the elastic Ig-tandem at 3.3Å resolution. The model reveals the molecular principles of Ig-arraying at the skeletal I-band of titin as mediated by conserved Ig-Ig transition motifs. Regular domain arrangements within this fragment point at the existence of a high-order in the fine structure of the filament, which is confirmed by EM data on a 19-mer poly-Ig segment. Our findings indicate a long-range, supra-order in the skeletal I-band of titin, where assembly of Ig domains into dynamical super-motifs is essential for the elastic function of the filament. We propose a novel model of spring mechanism for poly-Ig elasticity in titin based on a “carpenter ruler” model of skeletal I-band architecture. Furthermore, we have focused on the recruitment of the ubiquitin ligase MURF1 to the M-line signalosome through its specific interaction with titin domains A168 A170. MuRF1 contains several oligomerization motifs in succession, which indicates a possible need for tight regulation. We have therefore analyzed their influence on the oligomeric state of the protein. Our SEC-MALS data showed that the a-helical region of MuRF1 is dimeric in isolation, while in combination with the preceding B-Box domain, itself a dimerization motif, higher-order assembly is induced, which might be of physiological importance. We could also show that higher-order assembly of MuRF1 did not disrupt binding to A168-A170 in pull-down assays. Further biophysical or structural characterization of the complex of A168-A170 with MuRF1 constructs was hindered by the severely compromised solubility of the complex. Finally, we have successfully solved the crystal structure of the FnIII-Kin-Ig region of twitchin, which corresponds to titin A170-TK-M1. The N-terminal linker wraps around the kinase domain and positions the preceding FnIII domain in such a way that it blocks the autoregulatory tail in its inhibitory positon. Thus, from the structure we could conclude that stretch-activation of Twc kinase seems unlikely and instead propose phosphorylation of Y 104 as a possible activation mechanism. Our findings illustrate how the structural and functional diversity in titin’s modular architecture has evolved not only on the basis of individual domains. Rather, functionality often involves adaptation of several neighboring domains or even whole Ig tandems/super-repeats. This is reflected in variations in mechanical and dynamic properties observed in different parts of the chain and highlights the necessity of working with representative multi-domain fragments to gain a comprehensive understanding of the titin chai