Folding of Cu, Zn superoxide dismutase and Familial Amyotrophic Lateral Sclerosis

Cu,Zn superoxide dismutase (SOD1) has been implicated in the familial form of the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). It has been suggested that mutant mediated SOD1 misfolding/aggregation is an integral part of the pathology of ALS. We study the folding thermodynamics and kinetics of SOD1 using a hybrid molecular dynamics approach. We reproduce the experimentally observed SOD1 folding thermodynamics and find that the residues which contribute the most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly, we find that these residues are located on the surface of the protein and not in the hydrophobic core. Mutations in some of the identified residues are found in patients with the disease. We argue that the identified residues may play an important role in aggregation. To further characterize the folding of SOD1, we study the role of cysteine residues in folding and find that non-native disulfide bond formation may significantly alter SOD1 folding dynamics and aggregation propensity.Comment: 16 pages, 5 figure

Biophysical Mechanisms of Protein Aggregation

Protein aggregation related toxicity is implicated in a variety of neurodegenerative diseases including Alzheimer's, Huntington's, prion and Amyotrophic Lateral Sclerosis (ALS). The proteins or peptides known to aggregate in disease are unrelated in their amino acid sequence and native structure but form structurally similar aggregates - amyloids. Studies outlined in this dissertation were aimed at uncovering the underlying biophysical mechanisms of amyloid formation, and lay the groundwork to develop rational strategies to combat neurodegenerative diseases. More than 100 point mutations in the homodimeric metalloenzyme Cu, Zn superoxide (SOD1) dismutase are involved in a genetically inherited familial form of ALS (FALS). We have discovered a mechanism of in vitro SOD1 aggregation in which the native SOD1 dimer dissociates, metals are lost from the monomers and the resulting apo-monomers oligomerize in a rate-limiting step. Further, we have computationally estimated that a majority of FALS-associated point mutants in SOD1 (70 out of the 75 studied) decrease dimer stability and/or increase dimer dissociation propensity. Thus, we have proposed that the underlying biophysical basis of FALS-linked SOD1 aggregation is the mutation-induced increase in the propensity to form apo-monomers. To uncover the molecular determinants of SOD1 apo-monomer oligomerization, the rate-limiting step in aggregation, we have developed two complementary in silico approaches: (a) we have identified sequence fragments of SOD1 that have a high self-association propensity, and (b) we have performed molecular dynamics simulations of model SOD1 monomer and dimer folding and misfolding. In both cases, we have identified key residue-residue interactions in SOD1 responsible for maintaining fidelity to its native state. We have proposed that the disruption of one or more of these key interactions ("hot spots") is implicated in non-native oligomerization. To understand the effect of FALS mutations on the key interactions involved in maintaining native-state fidelity, we have studied the nanosecond dynamics of wild type SOD1 and 3 FALS-associated mutant apo-dimers and apo-monomers. We found that in wild type SOD1 the motions of the dimer interface are mechanically coupled to the motions of the structurally distal metal-coordinating loops of both monomeric subunits. We further found that the strain induced in the protein by dimer dissociation, point mutations, or by exposure to high temperature is transmitted to a specific hairpin in the protein, previously found to be implicated in maintaining fold fidelity. The altered dynamics of mutant SOD1 dimers and monomers provides structural insights into the flexibility required for oligomerization. Collectively, findings in this dissertation have enhanced our understanding of the complex mechanisms of protein aggregation. Mechanisms established and structural insights obtained herein may facilitate rational design of small molecules to prevent protein aggregation, hence provide a therapeutic intervention strategy in neurodegenerative diseases.Doctor of Philosoph

Comparative study of factors affecting productivity and cycle time of different excavators and their bucket size

Earthmoving and constructing equipments have evolved significantly during the past century. In every construction project some type of excavation must be performed. Excavators are primary earthmoving machines and equipment used to excavate earth and related materials. Contractors generally depend on their experience for selecting the right excavator for a job. Hence there is a need to build an understanding of how machine usage affects performance, extending across productivity. This study focuses on study of actual productivity against the theoretical productivity to demonstrate the loss of productivity. This real time monitoring of the heavy equipment can help practitioners improve machine intensive and cyclic earthmoving operations

De Novo Enzyme Design Using Rosetta3

The Rosetta de novo enzyme design protocol has been used to design enzyme catalysts for a variety of chemical reactions, and in principle can be applied to any arbitrary chemical reaction of interest, The process has four stages: 1) choice of a catalytic mechanism and corresponding minimal model active site, 2) identification of sites in a set of scaffold proteins where this minimal active site can be realized, 3) optimization of the identities of the surrounding residues for stabilizing interactions with the transition state and primary catalytic residues, and 4) evaluation and ranking the resulting designed sequences. Stages two through four of this process can be carried out with the Rosetta package, while stage one needs to be done externally. Here, we demonstrate how to carry out the Rosetta enzyme design protocol from start to end in detail using for illustration the triosephosphate isomerase reaction

Molecular Origin of Polyglutamine Aggregation in Neurodegenerative Diseases

Expansion of polyglutamine (polyQ) tracts in proteins results in protein aggregation and is associated with cell death in at least nine neurodegenerative diseases. Disease age of onset is correlated with the polyQ insert length above a critical value of 35–40 glutamines. The aggregation kinetics of isolated polyQ peptides in vitro also shows a similar critical-length dependence. While recent experimental work has provided considerable insights into polyQ aggregation, the molecular mechanism of aggregation is not well understood. Here, using computer simulations of isolated polyQ peptides, we show that a mechanism of aggregation is the conformational transition in a single polyQ peptide chain from random coil to a parallel β-helix. This transition occurs selectively in peptides longer than 37 glutamines. In the β-helices observed in simulations, all residues adopt β-strand backbone dihedral angles, and the polypeptide chain coils around a central helical axis with 18.5 ± 2 residues per turn. We also find that mutant polyQ peptides with proline-glycine inserts show formation of antiparallel β-hairpins in their ground state, in agreement with experiments. The lower stability of mutant β-helices explains their lower aggregation rates compared to wild type. Our results provide a molecular mechanism for polyQ-mediated aggregation

The Length Dependence of the PolyQ-mediated Protein Aggregation

Polyglutamine (polyQ) repeat disorders are caused by the expansion of CAG tracts in certain genes, resulting in transcription of proteins with abnormally long polyQ inserts. When these inserts expand beyond 35-45 glutamines, affected proteins form toxic aggregates, leading to neuron death. Chymotrypsin inhibitor 2 (CI2) with an inserted glutamine repeat has previously been used to model polyQ-mediated aggregation in vitro. However, polyQ insertion lengths in these studies have been kept below the pathogenic threshold. We perform molecular dynamics simulations to study monomer folding dynamics and dimer formation in CI2-polyQ chimeras with insertion lengths of up to 80 glutamines. Our model recapitulates the experimental results of previous studies of chimeric CI2 proteins, showing high folding cooperativity of monomers as well as protein association via domain swapping. Surprisingly, for chimeras with insertion lengths above the pathogenic threshold, monomer folding cooperativity decreases and the dominant mode for dimer formation becomes interglutamine hydrogen bonding. These results support a mechanism for pathogenic polyQ-mediated aggregation, in which expanded polyQ tracts destabilize affected proteins and promote the formation of partially unfolded intermediates. These unfolded intermediates form aggregates through associations by interglutamine interactions

De Novo Enzyme Design Using Rosetta3

The Rosetta de novo enzyme design protocol has been used to design enzyme catalysts for a variety of chemical reactions, and in principle can be applied to any arbitrary chemical reaction of interest, The process has four stages: 1) choice of a catalytic mechanism and corresponding minimal model active site, 2) identification of sites in a set of scaffold proteins where this minimal active site can be realized, 3) optimization of the identities of the surrounding residues for stabilizing interactions with the transition state and primary catalytic residues, and 4) evaluation and ranking the resulting designed sequences. Stages two through four of this process can be carried out with the Rosetta package, while stage one needs to be done externally. Here, we demonstrate how to carry out the Rosetta enzyme design protocol from start to end in detail using for illustration the triosephosphate isomerase reaction

Molecular Origin of Polyglutamine Aggregation in Neurodegenerative Diseases

Expansion of polyglutamine (polyQ) tracts in proteins results in protein aggregation and is associated with cell death in at least nine neurodegenerative diseases. Disease age of onset is correlated with the polyQ insert length above a critical value of 35–40 glutamines. The aggregation kinetics of isolated polyQ peptides in vitro also shows a similar critical-length dependence. While recent experimental work has provided considerable insights into polyQ aggregation, the molecular mechanism of aggregation is not well understood. Here, using computer simulations of isolated polyQ peptides, we show that a mechanism of aggregation is the conformational transition in a single polyQ peptide chain from random coil to a parallel β-helix. This transition occurs selectively in peptides longer than 37 glutamines. In the β-helices observed in simulations, all residues adopt β-strand backbone dihedral angles, and the polypeptide chain coils around a central helical axis with 18.5 ± 2 residues per turn. We also find that mutant polyQ peptides with proline-glycine inserts show formation of antiparallel β-hairpins in their ground state, in agreement with experiments. The lower stability of mutant β-helices explains their lower aggregation rates compared to wild type. Our results provide a molecular mechanism for polyQ-mediated aggregation

RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite

Macromolecular modeling and design are increasingly useful in basic research, biotechnology, and teaching. However, the absence of a user-friendly modeling framework that provides access to a wide range of modeling capabilities is hampering the wider adoption of computational methods by non-experts. RosettaScripts is an XML-like language for specifying modeling tasks in the Rosetta framework. RosettaScripts provides access to protocol-level functionalities, such as rigid-body docking and sequence redesign, and allows fast testing and deployment of complex protocols without need for modifying or recompiling the underlying C++ code. We illustrate these capabilities with RosettaScripts protocols for the stabilization of proteins, the generation of computationally constrained libraries for experimental selection of higher-affinity binding proteins, loop remodeling, small-molecule ligand docking, design of ligand-binding proteins, and specificity redesign in DNA-binding proteins

Introduction to the Rosetta Special Collection.

The Rosetta macromolecular modeling software is a versatile, rapidly developing set of tools that are now being routinely utilized to address state-of-the-art research challenges in academia and industrial research settings. A Rosetta Conference (RosettaCon) describing updates to the Rosetta source code is held annually. Every two years, a Rosetta Conference (RosettaCon) special collection describing the results presented at the annual conference by participating RosettaCommons labs is published by the Public Library of Science (PLOS). This is the introduction to the third RosettaCon 2014 Special Collection published by PLOS