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

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

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

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

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

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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

Can contact potentials reliably predict stability of proteins

The simplest approximation of interaction potential between amino acid residues in proteins is the contact potential, which defines the effective free energy of a protein conformation by a set of amino acid contacts formed in this conformation. Finding a contact potential capable of predicting free energies of protein states across a variety of protein families will aid protein folding and engineering in silico on a computationally tractable time-scale. We test the ability of contact potentials to accurately and transferably (across various protein families) predict stability changes of proteins upon mutations. We develop a new methodology to determine the contact potentials in proteins from experimental measurements of changes in protein’s thermodynamic stabilities ðDDGÞ upon mutations. We apply our methodology to derive sets of contact interaction parameters for a hierarchy of interaction models including solvation and multi-body contact parameters. We test how well our models reproduce experimental measurements by statistical tests. We evaluate the maximum accuracy of predictions obtained by using contact potentials and the correlation between parameters derived from different data-sets of experimental ðDDGÞ values. We argue that it is impossible to reach experimental accuracy and derive fully transferable contact parameters using the contact models of potentials. However, contact parameters may yield reliable predictions of DDG for datasets of mutations confined to the same amino acid positions in the sequence of a single protein