Applying graph theory to protein structures: an Atlas of coiled coils.

Motivation: To understand protein structure, folding and function fully and to design proteins de novo reliably, we must learn from natural protein structures that have been characterized experimentally. The number of protein structures available is large and growing exponentially, which makes this task challenging. Indeed, computational resources are becoming increasingly important for classifying and analyzing this resource. Here, we use tools from graph theory to define an Atlas classification scheme for automatically categorizing certain protein substructures. Results: Focusing on the α-helical coiled coils, which are ubiquitous protein-structure and protein-protein interaction motifs, we present a suite of computational resources designed for analyzing these assemblies. iSOCKET enables interactive analysis of side-chain packing within proteins to identify coiled coils automatically and with considerable user control. Applying a graph theory-based Atlas classification scheme to structures identified by iSOCKET gives the Atlas of Coiled Coils, a fully automated, updated overview of extant coiled coils. The utility of this approach is illustrated with the first formal classification of an emerging subclass of coiled coils called α-helical barrels. Furthermore, in the Atlas, the known coiled-coil universe is presented alongside a partial enumeration of the 'dark matter' of coiled-coil structures; i.e. those coiled-coil architectures that are theoretically possible but have not been observed to date, and thus present defined targets for protein design. Availability and implementation: iSOCKET is available as part of the open-source GitHub repository associated with this work (https://github.com/woolfson-group/isocket). This repository also contains all the data generated when classifying the protein graphs. The Atlas of Coiled Coils is available at: http://coiledcoils.chm.bris.ac.uk/atlas/app.

'Something in the way she moves': The functional significance of flexibility in the multiple roles of protein disulfide isomerase (PDI).

Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI.

ISAMBARD: an open-source computational environment for biomolecular analysis, modelling and design.

MOTIVATION: The rational design of biomolecules is becoming a reality. However, further computational tools are needed to facilitate and accelerate this, and to make it accessible to more users. RESULTS: Here we introduce ISAMBARD, a tool for structural analysis, model building and rational design of biomolecules. ISAMBARD is open-source, modular, computationally scalable and intuitive to use. These features allow non-experts to explore biomolecular design in silico. ISAMBARD addresses a standing issue in protein design, namely, how to introduce backbone variability in a controlled manner. This is achieved through the generalization of tools for parametric modelling, describing the overall shape of proteins geometrically, and without input from experimentally determined structures. This will allow backbone conformations for entire folds and assemblies not observed in nature to be generated de novo, that is, to access the 'dark matter of protein-fold space'. We anticipate that ISAMBARD will find broad applications in biomolecular design, biotechnology and synthetic biology. AVAILABILITY AND IMPLEMENTATION: A current stable build can be downloaded from the python package index (https://pypi.python.org/pypi/isambard/) with development builds available on GitHub (https://github.com/woolfson-group/) along with documentation, tutorial material and all the scripts used to generate the data described in this paper. CONTACT: d.n.woolfson@bristol.ac.uk or chris.wood@bristol.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Membrane-spanning α-helical barrels as tractable protein-design targets.

The rational (de novo) design of membrane-spanning proteins lags behind that for water-soluble globular proteins. This is due to gaps in our knowledge of membrane-protein structure, and experimental difficulties in studying such proteins compared to water-soluble counterparts. One limiting factor is the small number of experimentally determined three-dimensional structures for transmembrane proteins. By contrast, many tens of thousands of globular protein structures provide a rich source of 'scaffolds' for protein design, and the means to garner sequence-to-structure relationships to guide the design process. The α-helical coiled coil is a protein-structure element found in both globular and membrane proteins, where it cements a variety of helix-helix interactions and helical bundles. Our deep understanding of coiled coils has enabled a large number of successful de novo designs. For one class, the α-helical barrels-that is, symmetric bundles of five or more helices with central accessible channels-there are both water-soluble and membrane-spanning examples. Recent computational designs of water-soluble α-helical barrels with five to seven helices have advanced the design field considerably. Here we identify and classify analogous and more complicated membrane-spanning α-helical barrels from the Protein Data Bank. These provide tantalizing but tractable targets for protein engineering and de novo protein design.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

Engineering protein stability with atomic precision in a monomeric miniprotein.

Miniproteins simplify the protein-folding problem, allowing the dissection of forces that stabilize protein structures. Here we describe PPα-Tyr, a designed peptide comprising an α-helix buttressed by a polyproline II helix. PPα-Tyr is water soluble and monomeric, and it unfolds cooperatively with a midpoint unfolding temperature (TM) of 39 °C. NMR structures of PPα-Tyr reveal proline residues docked between tyrosine side chains, as designed. The stability of PPα is sensitive to modifications in the aromatic residues: replacing tyrosine with phenylalanine, i.e., changing three solvent-exposed hydroxyl groups to protons, reduces the TM to 20 °C. We attribute this result to the loss of CH-π interactions between the aromatic and proline rings, which we probe by substituting the aromatic residues with nonproteinogenic side chains. In analyses of natural protein structures, we find a preference for proline-tyrosine interactions over other proline-containing pairs, and observe abundant CH-π interactions in biologically important complexes between proline-rich ligands and SH3 and similar domains.

De novo protein design: how do we expand into the universe of possible protein structures?

Protein scientists are paving the way to a new phase in protein design and engineering. Approaches and methods are being developed that could allow the design of proteins beyond the confines of natural protein structures. This possibility of designing entirely new proteins opens new questions: What do we build? How do we build into protein-structure space where there are few, if any, natural structures to guide us? To what uses can the resulting proteins be put? And, what, if anything, does this pursuit tell us about how natural proteins fold, function and evolve? We describe the origins of this emerging area of fully de novo protein design, how it could be developed, where it might lead, and what challenges lie ahead.

Characterization of folding cores in the cyclophilin A-cyclosporin A complex.

Determining the folding core of a protein yields information about its folding process and dynamics. The experimental procedures for identifying the amino acids that make up the folding core include hydrogen-deuterium exchange and Φ-value analysis and can be expensive and time consuming. Because of this, there is a desire to improve upon existing methods for determining protein folding cores theoretically. We have obtained HDX data for the complex of cyclophilin A with the immunosuppressant cyclosporin A. We compare these data, as well as literature values for uncomplexed cyclophilin A, to theoretical predictions using a combination of rigidity analysis and coarse-grained simulations of protein motion. We find that in this case, the most specific prediction of folding cores comes from a combined approach that models the rigidity of the protein using the first software suite and the dynamics of the protein using the froda tool.

Does deamidation cause protein unfolding? A top-down tandem mass spectrometry study.

Deamidation is a nonenzymatic post-translational modification of asparagine to aspartic acid or glutamine to glutamic acid, converting an uncharged amino acid to a negatively charged residue. It is plausible that deamidation of asparagine and glutamine residues would result in disruption of a proteins' hydrogen bonding network and thus lead to protein unfolding. To test this hypothesis Calmodulin and B2M were deamidated and analyzed using tandem mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The gas phase hydrogen bonding networks of deamidated and nondeamidated protein isoforms were probed by varying the infra-red multi-photon dissociation laser power in a linear fashion and plotting the resulting electron capture dissociation fragment intensities as a melting curve at each amino acid residue. Analysis of the unfolding maps highlighted increased fragmentation at lower laser powers localized around heavily deamidated regions of the proteins. In addition fragment intensities were decreased across the rest of the proteins which we propose is because of the formation of salt-bridges strengthening the intramolecular interactions of the central regions. These results were supported by a computational flexibility analysis of the mutant and unmodified proteins, which would suggest that deamidation can affect the global structure of a protein via modification of the hydrogen bonding network near the deamidation site and that top down FTICR-MS is an appropriate technique for studying protein folding.