RosettaSurf—A surface-centric computational design approach

Proteins are typically represented by discrete atomic coordinates providing an accessible framework to describe different conformations. However, in some fields proteins are more accurately represented as near-continuous surfaces, as these are imprinted with geometric (shape) and chemical (electrostatics) features of the underlying protein structure. Protein surfaces are dependent on their chemical composition and, ultimately determine protein function, acting as the interface that engages in interactions with other molecules. In the past, such representations were utilized to compare protein structures on global and local scales and have shed light on functional properties of proteins. Here we describe RosettaSurf, a surface-centric computational design protocol, that focuses on the molecular surface shape and electrostatic properties as means for protein engineering, offering a unique approach for the design of proteins and their functions. The RosettaSurf protocol combines the explicit optimization of molecular surface features with a global scoring function during the sequence design process, diverging from the typical design approaches that rely solely on an energy scoring function. With this computational approach, we attempt to address a fundamental problem in protein design related to the design of functional sites in proteins, even when structurally similar templates are absent in the characterized structural repertoire. Surface-centric design exploits the premise that molecular surfaces are, to a certain extent, independent of the underlying sequence and backbone configuration, meaning that different sequences in different proteins may present similar surfaces. We benchmarked RosettaSurf on various sequence recovery datasets and showcased its design capabilities by generating epitope mimics that were biochemically validated. Overall, our results indicate that the explicit optimization of surface features may lead to new routes for the design of functional proteins.     

Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents

Background

Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur.

Methods

Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone.

Results

Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde–dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes.

Conclusions and general significance

Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents.     

Some aspects of segment formation and post-placental development in Peripatus acacioi marcus and marcus (Onychophora)

The surface morphology of the anterior-to-posterior sequence of segment formation in embryos of a viviparous neotropical onychophoran and aspects of post-placental development seen using scanning electron microscopy are described. When all the segments have formed and the walking legs have completed their elongation, the body surface becomes covered with an embryonic cuticle that does not exhibit the hydrofuge properties seen in the adult cuticle. As soon as the walking legs have reached their full length, barbed projections are formed at their distal extremities. These projections are extensions of single cells and are covered by the embryonic cuticle. Transmission electron microscopy reveals that the cells at the distal ends of the legs and their projections have many pinocytotic vesicles at their surfaces. The cytoplasm of these cells and their projections is rich in mitochondria, rough endoplasmic reticulum, glycogen, and granules of storage material. There are minor differences in the surface morphology of the projections found at the ends of the walking legs in embryos of Peripatus acacioi and those of Peripatus biolleyi. The projections and the embryonic cuticle persist thoughout postplacental development. The role of the projections in the uptake of material by the embryo from the uterus is discussed and the possible phylogenetic significance of these projections is suggested.     

Correlation of structure to function of the luminal cell surface in the uterine epithelium of mouse and man

Summary The electron microscopical appearance of the luminal cell surface of the uterine epithelium in mouse and man was similar at similar stages of the functional activity of the epithelial cells. The inactive stage was characterized by 0.1 long microvilli, the stage of the sperm passage at the time of ovulation was characterized by 1 long microvilli, and that of the egg implantation by an irregular cell surface with several large projections.The similarity of structural changes in the two species might imply a basic function of the cell membrane in uterine physiology. The morphological changes of the cell membrane indicate that its physical or chemical properties might be changed during the different functional states of the cell.Supported by a grant from Stifteken Therese och Johan Anderssons Minne.     

Scanning electron microscopy of the respiratory surfaces of Saccobranchus (=Heteropneustes) fossilis (Bloch)

Summary The gill secondary lamellae are generally covered with epithelial cells whose outer surfaces form numerous microvilli. The surface of the primary lamellae is characterised by microridges. A particular type of surface sculpturing seems to be associated with given cell boundaries.Further evidence for the derivation of the air tube and fans which guard its entrance by modification of the basic gill structure has been obtained from both the gross surface architecture and microstructure of the individual cell surfaces. Secondary lamellae are represented by stubby projections which generally have a biserial arrangement. The outer surfaces of the epithelia overlying the capillaries of these respiratory islets are coated with microvilli as in the secondary lamellae. On the other hand, the relatively smooth-surfaced lanes between groups of respiratory islets have a microridged surface similar to that of the primary gill lamellae.It is suggested that previous estimates of surface area, and consequently diffusing capacities of the air-breathing organ, have been low in view of the increased surface, due to both their gross and microstructure. Estimates for gill surface area may need very little correction as the spaces between the microvilli and microridges are probably filled with mucus under normal conditions.We thank Mr. John Clements for his excellent technical assistance and the Department of Botany, Bristol University for the use of their scanning electron microscope     

Macromolecular solvation energies derived from small molecule crystal morphology.

The morphology of small molecule crystals provides a model for evaluating surface solvation energies in a system with similar packing density to that observed for amino acid residues in proteins. The solvation energies associated with the transfer of methylene and carboxyl groups between vacuum and aqueous phases are estimated to be approx. $40 and -260 cal/A2, respectively, from an analysis of the morphology of succinic acid crystals. These solvation energies predict values for contact angles in reasonable agreement with measurements determined from macroscopic monolayer surfaces. Transfer free energies between vapor and water phases for a series of carboxylic acids are also predicted reasonably well by these solvation energies, provided the surface exposure of different groups is quantitated with the molecular surface area rather than the more traditional accessible surface area. In general, molecular surfaces and molecular surface areas are seen to have important advantages for characterizing the structure and energetics of macromolecular surfaces. Crystal faces of succinic acid with the lowest surface energies in aqueous solution are characteristically smooth. Increasing surface roughness and apolarity are associated with higher surface energies, which suggests an approach for modifying the surface properties of proteins and other macromolecules.     

Accuracy of functional surfaces on comparatively modeled protein structures

Identification and characterization of protein functional surfaces are important for predicting protein function, understanding enzyme mechanism, and docking small compounds to proteins. As the rapid speed of accumulation of protein sequence information far exceeds that of structures, constructing accurate models of protein functional surfaces and identify their key elements become increasingly important. A promising approach is to build comparative models from sequences using known structural templates such as those obtained from structural genome projects. Here we assess how well this approach works in modeling binding surfaces. By systematically building three-dimensional comparative models of proteins using Modeller, we determine how well functional surfaces can be accurately reproduced. We use an alpha shape based pocket algorithm to compute all pockets on the modeled structures, and conduct a large-scale computation of similarity measurements (pocket RMSD and fraction of functional atoms captured) for 26,590 modeled enzyme protein structures. Overall, we find that when the sequence fragment of the binding surfaces has more than 45% identity to that of the template protein, the modeled surfaces have on average an RMSD of 0.5 Å, and contain 48% or more of the binding surface atoms, with nearly all of the important atoms in the signatures of binding pockets captured.     

Discovery of similar regions on protein surfaces.

Discovery of a similar region on two protein surfaces can lead to important inference about the functional role or molecular interaction of this region for one of the proteins if such information is available for the other. We propose a new characterization of protein surfaces based on a spin-image representation of the surfaces that facilitates the simultaneous search of the entire surface of each of two proteins for a matching region. For a surface point, we introduce spin-image profiles that are related to the degree of exposure of the point to identify structurally equivalent surface regions in two proteins. Unlike some related methods, we do not assume that a known fixed region of one of the protein surfaces is to be matched on the other protein surface. Rather, we search for the largest similar regions on each of the two surfaces. In spite of the fact that this approach is entirely geometric and no use is made of physicochemical properties of the protein surfaces or fold information, it is effective in identifying similar regions on both surfaces even when the region corresponds to a binding site on one of the proteins. The discovery of similar regions on two or more proteins also has implications for drug design and pharmacophore identification. We present experimental results from datasets of more than 50 protein surfaces.     

Protein Functional Surfaces: Global Shape Matching and Local Spatial Alignments of Ligand Binding Sites

Background

Protein surfaces comprise only a fraction of the total residues but are the most conserved functional features of proteins. Surfaces performing identical functions are found in proteins absent of any sequence or fold similarity. While biochemical activity can be attributed to a few key residues, the broader surrounding environment plays an equally important role.

Results

We describe a methodology that attempts to optimize two components, global shape and local physicochemical texture, for evaluating the similarity between a pair of surfaces. Surface shape similarity is assessed using a three-dimensional object recognition algorithm and physicochemical texture similarity is assessed through a spatial alignment of conserved residues between the surfaces. The comparisons are used in tandem to efficiently search the Global Protein Surface Survey (GPSS), a library of annotated surfaces derived from structures in the PDB, for studying evolutionary relationships and uncovering novel similarities between proteins.

Conclusion

We provide an assessment of our method using library retrieval experiments for identifying functionally homologous surfaces binding different ligands, functionally diverse surfaces binding the same ligand, and binding surfaces of ubiquitous and conformationally flexible ligands. Results using surface similarity to predict function for proteins of unknown function are reported. Additionally, an automated analysis of the ATP binding surface landscape is presented to provide insight into the correlation between surface similarity and function for structures in the PDB and for the subset of protein kinases.     

Surface specializations of the epithelial cells at the tip of the optic tentacle,dorsal surface of the head and ventral surface of the foot in Helix aspersa

Summary Morphologically the surface specializations of the epithelium covering the dorsal head and ventral foot regions in Helix aspersa consists either of cilia or microvilli respectively. The epithelium at the tip of the optic tentacle is a simple one. Each epithelial cell has a number of cilia-like projections from their free surfaces. These projections usually branch at their tips into two or three slender, microvilli-like structures. From the bases of the cilia-like projections arise numerous, tubular processes which form a thick, spongy layer interspersed between these projections. The microvilli-like structures are immersed in a fine, fibrous mat; unlike the fibrous mats on the dorsal head and ventral foot epithelia this material does not autofluoresce. It is suggested that it arises from the collar cells and not from typical mucocytes. The functional relationship between these surface specializations of the optic tentacle epithelium and the abundance of sensory axons in this region is discussed. These epithelial cell projections on the tentacle probably function not only as a protective covering but also to create a fluid trap for odours in the ambient air. The various contacts between epithelial cells serve to maintain the integrity of the epithelium while allowing for stretching due to protrusion of the tentacle.This work has been supported by the Australian Research Grants Committee.     

Elaborations of the basal surface of the cells of the Malpighian tubules of an insect

Electrical measurements of membrane potential and resistance using intracellular microelectrodes showed that the fluid-secreting parts of the Malpighian tubules of Rhodnius have superficial regions different in electrical properties from the main body of the cell. The membrane potential in these superficial regions was smaller by 30-40 mV, but showed a standard depolarization on changing the potassium concentration of the bathing medium. The response to changes in the external chloride concentration also differed in the two regions, a finding that was reinforced by different responses to the drug, furosemide. Electron microscopy of the basal regions of the cells revealed many long cellular projections that run parallel to the cell surface and interdigitate with similar projections from neighbouring cells. The degree of interdigitation was examined by marking individual cells with alcian blue or by horseradish peroxidase injection. A survey of the published micrographs of insect Malpighian tubules shows that most have similar projections on their basal surfaces and not the simple basal infoldings previously supposed.     

SURF’s UP! — Protein classification by surface comparisons

Large-scale genome sequencing and structural genomics projects generate numerous sequences and structures for 'hypothetical' proteins without functional characterizations. Detection of homology to experimentally characterized proteins can provide functional clues, but the accuracy of homology-based predictions is limited by the paucity of tools for quantitative comparison of diverging residues responsible for the functional divergence. SURF'S UP! is a web server for analysis of functional relationships in protein families, as inferred from protein surface maps comparison according to the algorithm. It assigns a numerical score to the similarity between patterns of physicochemical features(charge, hydrophobicity) on compared protein surfaces. It allows recognizing clusters of proteins that have similar surfaces, hence presumably similar functions. The server takes as an input a set of protein coordinates and returns files with "spherical coordinates" of proteins in a PDB format and their graphical presentation, a matrix with values of mutual similarities between the surfaces, and the unrooted tree that represents the clustering of similar surfaces, calculated by the neighbor-joining method. SURF'S UP! facilitates the comparative analysis of physicochemical features of the surface, which are the key determinants of the protein function. By concentrating on coarse surface features, SURF'S UP! can work with models obtained from comparative modelling. Although it is designed to analyse the conservation among homologs, it can also be used to compare surfaces of non-homologous proteins with different three-dimensional folds, as long as a functionally meaningful structural superposition is supplied by the user. Another valuable characteristic of our method is the lack of initial assumptions about the functional features to be compared. SURF'S UP! is freely available for academic researchers at http://asia.genesilico.pl/surfs_up/.     

Comparative sequence and structure analysis of eIF1A and eIF1AD

Background

Eukaryotic translation initiation factor 1A (eIF1A) is universally conserved in all organisms. It has multiple functions in translation initiation, including assembly of the ribosomal pre-initiation complexes, mRNA binding, scanning, and ribosomal subunit joining. eIF1A binds directly to the small ribosomal subunit, as well as to several other translation initiation factors. The structure of an eIF1A homolog, the eIF1A domain-containing protein (eIF1AD) was recently determined but its biological functions are unknown. Since eIF1AD has a known structure, as well as a homolog, whose structure and functions have been extensively studied, it is a very attractive target for sequence and structure analysis.

Results

Structure/sequence analysis of eIF1AD found significant conservation in the surfaces corresponding to the ribosome-binding surfaces of its paralog eIF1A, including a nearly invariant surface-exposed tryptophan residue, which plays an important role in the interaction of eIF1A with the ribosome. These results indicate that eIF1AD may bind to the ribosome, similar to its paralog eIF1A, and could have roles in ribosome biogenenesis or regulation of translation. We identified conserved surfaces and sequence motifs in the folded domain as well as the C-terminal tail of eIF1AD, which are likely protein-protein interaction sites. The roles of these regions for eIF1AD function remain to be determined. We have also identified a set of trypanosomatid-specific surface determinants in eIF1A that could be a promising target for development of treatments against these parasites.

Conclusions

The results described here identify regions in eIF1A and eIF1AD that are likely to play major functional roles and are promising therapeutic targets. Our findings and hypotheses will promote new research and help elucidate the functions of eIF1AD.

     

The role of bacterial surface and substratum hydrophobicity in adhesion ofLeptospira biflexa serovarpatoc 1 to inert surfaces

Adhesion of the hydrophilicLeptospira biflexa serovarpatoc 1 (L. patoc) was consistently greater on inert hydrophobic surfaces than on hydrophilic surfaces (glass and plastic). When inert substrata were coated with fetal calf serum (FCS) or bovine serum albumin fraction V (BSA), however, surface hydrophobicity was reduced compared to untreated surfaces, but adhesion ofL. patoc increased. The mechanism of adhesion at protein-coated surfaces is likely to be different than that at untreated surfaces, but it is suggested that the adhesion is nonspecific, as the level of adhesion is similar for different protein coatings. Increased adhesion to FCS- and BSA-coated surfaces was apparently not associated with substrate utilization (scavenging of fatty acids) from the coatings, as essentially fatty acid-free BSA-coated surfaces had similar levels of adhesion. The presence of FCS in the diluent lowered the adhesion ofL. patoc regardless of the original nature of the substratum. This may result from the mutual repulsion of the bacterium and the substratum caused by the exclusion volumes of similar macromolecules adsorbed to both surfaces from the FCS solution.     

Understanding 2D projections on mirrors and on windows

Representational art tries to capture a 3D world on a 2D surface, and artists often discuss this in relation to the projected image on window panes and mirrors. But are 2D projections on transparent surfaces useful to learn about projections in general? Most people are unaware of the 2D projected size of objects on the surface of mirrors. They also incorrectly expect that these projections always get smaller with distance of the target object from the mirror, and do not change with distance of the observer (when the target is stationary). In this paper we extend this result about surfaces of mirrors to surfaces of windows, and we confirm that the errors that people make are not specific to Western culture by replicating the study in China. In contrast to their errors about projections, people are more accurate at predicting how field of view will vary depending on distance of the observer from a mirror or window. To explain how this pattern of (false) beliefs can stem from experience we argue that people do not perceive projections on transparent surfaces.     

Interactive computer surface graphic study of the binding of an antibody to the chromatin subunit

The binding of an IgG molecule to a chromatin subunit has been simulated by the interactive computer surface graphics technique. This technique permits the facile display of the surface of macromolecules for which atomic co-ordinates are known. The computer generated projections reveal spatial relationship between the IgG and the nucleosome. Studies of these projections provide insights into the immunogenicity and antigenicity of the chromatin subunit. The concepts discussed are helpful in understanding the manner in which IgG molecules specific to DNA bases, chromosomal proteins, irradiated DNA and to chemical carcinogens bind to the genome.     

Validation of a Laboratory Method for Evaluating Dynamic Properties of Reconstructed Equine Racetrack Surfaces

Background

Racetrack surface is a risk factor for racehorse injuries and fatalities. Current research indicates that race surface mechanical properties may be influenced by material composition, moisture content, temperature, and maintenance. Race surface mechanical testing in a controlled laboratory setting would allow for objective evaluation of dynamic properties of surface and factors that affect surface behavior.

Objective

To develop a method for reconstruction of race surfaces in the laboratory and validate the method by comparison with racetrack measurements of dynamic surface properties.

Methods

Track-testing device (TTD) impact tests were conducted to simulate equine hoof impact on dirt and synthetic race surfaces; tests were performed both in situ (racetrack) and using laboratory reconstructions of harvested surface materials. Clegg Hammer in situ measurements were used to guide surface reconstruction in the laboratory. Dynamic surface properties were compared between in situ and laboratory settings. Relationships between racetrack TTD and Clegg Hammer measurements were analyzed using stepwise multiple linear regression.

Results

Most dynamic surface property setting differences (racetrack-laboratory) were small relative to surface material type differences (dirt-synthetic). Clegg Hammer measurements were more strongly correlated with TTD measurements on the synthetic surface than the dirt surface. On the dirt surface, Clegg Hammer decelerations were negatively correlated with TTD forces.

Conclusions

Laboratory reconstruction of racetrack surfaces guided by Clegg Hammer measurements yielded TTD impact measurements similar to in situ values. The negative correlation between TTD and Clegg Hammer measurements confirms the importance of instrument mass when drawing conclusions from testing results. Lighter impact devices may be less appropriate for assessing dynamic surface properties compared to testing equipment designed to simulate hoof impact (TTD).

Potential Relevance

Dynamic impact properties of race surfaces can be evaluated in a laboratory setting, allowing for further study of factors affecting surface behavior under controlled conditions.     

Principles of nanostructure design with protein building blocks

Currently there is increasing interest in nanostructures and their design. Nanostructure design involves the ability to predictably manipulate the properties of the self-assembly of autonomous units. Autonomous units have preferred conformational states. The units can be synthetic material science-based or derived from functional biological macromolecules. Autonomous biological building blocks with available structures provide an extremely rich and useful resource for design. For proteins, the structural databases contain large libraries of protein molecules and their building blocks with a range of shapes, surfaces, and chemical properties. The introduction of engineered synthetic residues or short peptides into these can expand the available chemical space and enhance the desired properties. Here we focus on the principles of nanostructure design with protein building blocks.     

NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex.

We have isolated a new gene, NUP2, that encodes a constituent of the yeast-nuclear pore complex (NPC). The NUP2 protein sequence shares a central repetitive domain with NSP1 and NUP1, the two previously characterized yeast nucleoporins. Like NUP1 and NSP1, NUP2 localizes to discrete spots in the nuclear envelope, as determined by indirect immunofluorescence. Although the sequence similarity among these three nucleoporins suggests that they have a similar role in the nuclear pore complex, NUP2, in contrast to NSP1 and NUP1, is not required for growth. Some combinations of mutant alleles of NUP1, NSP1, and NUP2 display "synthetic lethal" relationships that provide evidence for functional interaction between these NPC components. This genetic evidence of overlapping function suggests that the nucleoporins act in concert, perhaps participating in the same step of the recognition or transit of macromolecules through the NPC.