nils-herrmann/clean_scirepeval huggingface dataset:
Unremodeled endochondral bone is a major architectural component of the cortical bone of the rat (Rattus norvegicus).
Venue: Journal of structural biology

The laboratory rat is one of the most frequently-used animal models for studying bone biology and skeletal diseases. Here we show that a substantial portion of the cortical bone of mature rats is primary endochondral bone, consisting of a disorganized arrangement of mineralized collagen fibers. We characterize the structure and mechanical properties of the cortical bone of the rat. We show that the cortical bone consists of two architecturally distinct regions. One region, consisting of well-organized circumferential lamellae (CLB), is located in the endosteal and/or the periosteal regions while another, disorganized region, is located in the more central region of the cortex. Unexpectedly, we found that the disorganized region contains many islands of highly mineralized cartilage. Micro tomography showed different structural and compositional properties of the two primary structural elements; the CLB region has lower mineral density, lower porosity, larger but fewer blood vessels and fewer lacunae. However, no difference was found in the average lacunar volume. Additionally the mean indentation modulus of the CLB region was lower than that of the disorganized region. The islands of calcified cartilage were found to be extremely stiff, with an indentation modulus of 33.4 ± 3.5GPa. We conclude that though the cortical bone of rats is in part lamellar, its architecture is markedly different from that of the cortical bone of humans, a fact that must be borne in mind when using the rat as a model animal for studies of human bone biology and disease.
On the function of chitin synthase extracellular domains in biomineralization.
Venue: Journal of structural biology

Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface.
Of mice, rats and men: trabecular bone architecture in mammals scales to body mass with negative allometry.
Venue: Journal of structural biology

Body mass (BM) in mammal species spans over six orders of magnitude. Although trabecular bone contributes to the mechanical properties of bones, we know much less about how trabecular bone scales with BM than about how cortical bone scales with BM. We therefore conducted a meta-analysis of the existing literature to test in rodents, humans and other mammals, predicted scaling properties between BM and several trabecular parameters: bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density (ConnD) and degree of anisotropy (DA). Our results show that BV/TV and DA are independent of BM and that Tb.N, Tb.Th and Tb.Sp scale with negative allometry relative to BM. Rodents appear to have relatively thicker and fewer trabeculae than humans, and we propose it that is due to a minimum thickness threshold "imposed" on mechanically functional trabeculae. Consequently, rodents (mice and rats) and humans demonstrate two distinct mechanisms to achieve variations in BV/TV. Although Tb.Th variation is the main contributing factor for differences in BV/TV in humans, Tb.N variation is the main contributing factor for differences in BV/TV in rodents. Our results also demonstrate no correlation between Tb.N and Tb.Th within each taxon (mice, rats and humans). Since rodents are a common animal model for research on bone biomechanics, the evidence that trabecular bone parameters scale and correlate differently in rodents than in humans suggests that care should be applied when extrapolating bone biomechanical results from small animals to large-bodied humans.
The challenge of determining handedness in electron tomography and the use of DNA origami gold nanoparticle helices as molecular standards.
Venue: Journal of structural biology

The apparent handedness of an EM-tomography reconstruction depends on a number of conventions and can be confused in many ways. As the number of different hardware and software combinations being used for electron tomography continue to climb, and the reconstructions being produced reach higher and higher resolutions, the need to verify the hand of the results has increased. Here we enumerate various steps in a typical tomography experiment that affect handedness and show that DNA origami gold nanoparticle helices can be used as convenient and fail-safe handedness standards.
A new family of proteins related to the HEAT-like repeat DNA glycosylases with affinity for branched DNA structures.
Venue: Journal of structural biology

The recently discovered HEAT-like repeat (HLR) DNA glycosylase superfamily is widely distributed in all domains of life. The present bioinformatics and phylogenetic analysis shows that HLR DNA glycosylase superfamily members in the genus Bacillus form three subfamilies: AlkC, AlkD and AlkF/AlkG. The crystal structure of AlkF shows structural similarity with the DNA glycosylases AlkC and AlkD, however neither AlkF nor AlkG display any DNA glycosylase activity. Instead, both proteins have affinity to branched DNA structures such as three-way and Holliday junctions. A unique β-hairpin in the AlkF/AlkG subfamily is most likely inserted into the DNA major groove, and could be a structural determinant regulating DNA substrate affinity. We conclude that AlkF and AlkG represent a new family of HLR proteins with affinity for branched DNA structures.
Design of net-charged abc-type collagen heterotrimers.
Venue: Journal of structural biology

Net-negatively-charged heterospecific A:B:C collagen peptide heterotrimers were designed using an automated computational approach. The design algorithm considers both target stability and the energy gap between the target states and misfolded competing states. Structural characterization indicates the net-negative charge balance on the new designs enhances the specificity of the target state at the expense of its stability.
Variations in the stability of NCR ene reductase by rational enzyme loop modulation.
Venue: Journal of structural biology

The engineering of protein stability is of major importance for the application of enzymes in a wide range of industrial applications. Here we study the determinants of the thermo- and solvent stability of the Zymomonas mobilis ene reductase NCR using a rational protein engineering approach based on analyses of structural and sequence data. We designed and created two loop mutants with the aim to increase their overall stability. They all retained catalytic activity but exhibited altered thermostability relative to the wild-type enzyme. The modulation of one specific loop segment near the active site of NCR showed an increased tolerance to organic solvents along with an enhanced thermostability.
Membrane curvature in flaviviruses.
Venue: Journal of structural biology

Coordinated interplay between membrane proteins and the lipid bilayer is required for such processes as transporter function and the entrance of enveloped viruses into host cells. In this study, three-dimensional cryo-electron microscopy density maps of mature and immature flaviviruses were analyzed to assess the curvature of the membrane leaflets and its relation to membrane-bound viral glycoproteins. The overall morphology of the viral membrane is determined by the icosahedral scaffold composed of envelope (E) and membrane (M) proteins through interaction of the proteins' stem-anchor regions with the membrane. In localized regions, small membrane areas exhibit convex, concave, flat or saddle-shaped surfaces that are constrained by the specific protein organization within each membrane leaflet. These results suggest that the organization of membrane proteins in small enveloped viruses mediate the formation of membrane curvature.
In vitro models of collagen biomineralization.
Venue: Journal of structural biology

Over the last several years, significant progress has been made toward understanding the mechanisms involved in the mineralization of hard collagenous tissues, such as bone and dentin. Particularly notable are the identification of transient mineral phases that are precursors to carbonated hydroxyapatite, the identification and characterization of non-collagenous proteins that are involved in controlling mineralization, and significant improvements in our understanding of the structure of collagen. These advances not only represent a paradigm shift in the way collagen mineralization is viewed and understood, but have also brought new challenges to light. In this review, we discuss how recent in vitro models have addressed critical questions regarding the role of the non-collagenous proteins in controlling mineralization, the nature of the interactions between amorphous calcium phosphate and collagen during the early stages of mineralization, and the role of collagen in the mineralization process. We discuss the significance of these findings in expanding our understanding of collagen biomineralization, while addressing some of the limitations that are inherent to in vitro systems.
Inelastic deformation and microcracking process in human dentin.
Venue: Journal of structural biology

Dentin is a mineralized collagen tissue with robust mechanical performance. Understanding the mechanical behavior of dentin and its relations to the dentinal structure can provides insight into the design strategies to achieve tooth functions. This study focuses on the inelastic deformation of human dentin and its underlying mechanisms. By combining four-point bending tests with fluorescent staining and laser scanning confocal microscopy, it was found that human dentin, especially root dentin, exhibited significant inelastic deformation and developed extensive microdamage in the form of microcracks prior to fracture. Dense and wavy microcracks spread uniformly across the tensile surface of root dentin, while compressive microcracks formed cross-hatched patterns. The presence of peritubular dentin in coronal dentin dramatically decreased the extent of microcracking, reducing inelasticity. Dentinal tubules were found to be initiation sites of both tensile and compressive microcracks. A unique crack propagation process was observed in root dentin under tension: numerous ring-shaped cracks formed at each dentinal tubule ahead of a growing crack tip. The advance of the tensile microcracks occurred by the merging of those ring-shaped cracks. The current findings on the microcracking process associated with inelastic deformation helps to understand the nature of strength and toughness in dentin, as well as the mechanical significance for structural variations across the whole tooth.
SM30 protein function during sea urchin larval spicule formation.
Venue: Journal of structural biology

A central issue in better understanding the process of biomineralization is to elucidate the function of occluded matrix proteins present in mineralized tissues. A potent approach to addressing this issue utilizes specific inhibitors of expression of known genes. Application of antisense oligonucleotides that specifically suppress translation of a given mRNA are capable of causing aberrant biomineralization, thereby revealing, at least in part, a likely function of the protein and gene under investigation. We have applied this approach to study the possible function(s) of the SM30 family of proteins, which are found in spicules, teeth, spines, and tests of Strongylocentrotus purpuratus as well as other euechinoid sea urchins. It is possible using the anti-SM30 morpholino-oligonucleotides (MO's) to reduce the level of these proteins to very low levels, yet the development of skeletal spicules in the embryo shows little or no aberration. This surprising result requires re-thinking about the role of these, and possibly other occluded matrix proteins.
Crystallization of bi-functional ligand protein complexes.
Venue: Journal of structural biology

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects.
Localization and quantitative co-localization of enamelin with amelogenin.
Venue: Journal of structural biology

Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1-8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 μm height, 33 μm width) and small (7 μm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.
Ligand promiscuity within the internal cavity of Epiphyas postvittana Takeout 1 protein.
Venue: Journal of structural biology

Takeout proteins are found across a diverse range of insect species and are thought to be involved in important aspects of insect physiology and behavior. These proteins act as ligand carriers, but the nature of their endogenous ligands remains unknown. The crystal structure of Epiphyas postvittana Takeout 1 (EpTo1), the only structure for any Takeout protein to date, revealed an α/β-wrap fold with a purely hydrophobic internal cavity of tubular shape. When recombinantly expressed in Escherichia coli, we previously showed that a surrogate ubiquinone-8 ligand binds within the internal cavity of EpTo1 with excellent shape complementarity. We have now expressed EpTo1 in an insect cell expression system devoid of ubiquinone-8, and solved its crystal structure at 2.2Å resolution. Using combined information from crystallography and mass spectrometry, we identify a mixture of fatty acid moieties, mostly myristic and palmitic acid, bound inside the EpTo1 cavity, mimicking the structure of the longer ubiquinone-8 compound. No significant alteration of the internal cavity was observed regardless of the bound ligands, ubiquinone-8 or fatty acids, suggesting that the internal cavity of EpTo1 forms a rigid scaffold that imposes strict structural constraints for selectivity and specificity of ligand(s) in vivo.
Protein design for pathway engineering.
Venue: Journal of structural biology

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.
An accurate binding interaction model in de novo computational protein design of interactions: if you build it, they will bind.
Venue: Journal of structural biology

Computational protein design efforts aim to create novel proteins and functions in an automated manner and, in the process, these efforts shed light on the factors shaping natural proteins. The focus of these efforts has progressed from the interior of proteins to their surface and the design of functions, such as binding or catalysis. Here we examine progress in the development of robust methods for the computational design of non-natural interactions between proteins and molecular targets such as other proteins or small molecules. This problem is referred to as the de novo computational design of interactions. Recent successful efforts in de novo enzyme design and the de novo design of protein-protein interactions open a path towards solving this problem. We examine the common themes in these efforts, and review recent studies aimed at understanding the nature of successes and failures in the de novo computational design of interactions. While several approaches culminated in success, the use of a well-defined structural model for a specific binding interaction in particular has emerged as a key strategy for a successful design, and is therefore reviewed with special consideration.
The molecular structure within dislocations in Cannabis sativa fibres studied by polarised Raman microspectroscopy.
Venue: Journal of structural biology

Polarised Raman micrsospectroscopy was employed to study the molecular structure within dislocations (slip planes) in the cell walls of Hemp fibre cells (Cannabis sativa (L.)). It was found that the cellulose microfibrils within dislocations have a different orientation than in the surrounding cell wall, and that the cellulose in the transition zones between a large dislocation and the surrounding wall may have yet another orientation. Furthermore, cellulose orientation seemed to be less uniform within dislocations than in the surrounding cell wall.
Structure-guided engineering of Anticalins with improved binding behavior and biochemical characteristics for application in radio-immuno imaging and/or therapy.
Venue: Journal of structural biology

Modern strategies in radio-immuno therapy and in vivo imaging require robust, small, and specific ligand-binding proteins. In this context we have previously developed artificial lipocalins, so-called Anticalins, with high binding activity toward rare-earth metal-chelate complexes using combinatorial protein design. Here we describe further improvement of the Anticalin C26 via in vitro affinity maturation to yield CL31, which has a fourfold slower dissociation half-life above 2h. Also, we present the crystallographic analyses of both the initial and the improved Anticalin, providing insight into the molecular mechanism of chelated metal binding and the role of amino acid substitutions during the step-wise affinity maturation. Notably, one of the four structurally variable loops that form the ligand pocket in the lipocalin scaffold undergoes a significant conformational change from C26 to CL31, acting as a lid that closes over the accommodated metal-chelate ligand. A systematic mutational study indicated that further improvement of ligand affinity is difficult to achieve while providing clues on the contribution of relevant side chains in the engineered binding pocket. Unexpectedly, some of the amino acid replacements led to strong increases - more then 10-fold - in the yield of soluble protein from periplasmic secretion in Escherichia coli.