Nanomechanical strength mechanisms of hierarchical biological materials and tissues

Biological protein materials (BPMs), intriguing hierarchical structures formed by assembly of chemical building blocks, are crucial for critical functions of life. The structural details of BPMs are fascinating: They represent a combination of universally found motifs such as alpha-helices or beta-sheets with highly adapted protein structures such as cytoskeletal networks or spider silk nanocomposites. BPMs combine properties like strength and robustness, self-healing ability, adaptability, changeability, evolvability and others into multi-functional materials at a level unmatched in synthetic materials. The ability to achieve these properties depends critically on the particular traits of these materials, first and foremost their hierarchical architecture and seamless integration of material and structure, from nano to macro. Here, we provide a brief review of this field and outline new research directions, along with a review of recent research results in the development of structure-property relationships of biological protein materials exemplified in a study of vimentin intermediate filaments.     

Nanomechanical strength mechanisms of hierarchical biological materials and tissues

Biological protein materials (BPMs), intriguing hierarchical structures formed by assembly of chemical building blocks, are crucial for critical functions of life. The structural details of BPMs are fascinating: They represent a combination of universally found motifs such as α-helices or β-sheets with highly adapted protein structures such as cytoskeletal networks or spider silk nanocomposites. BPMs combine properties like strength and robustness, self-healing ability, adaptability, changeability, evolvability and others into multi-functional materials at a level unmatched in synthetic materials. The ability to achieve these properties depends critically on the particular traits of these materials, first and foremost their hierarchical architecture and seamless integration of material and structure, from nano to macro. Here, we provide a brief review of this field and outline new research directions, along with a review of recent research results in the development of structure–property relationships of biological protein materials exemplified in a study of vimentin intermediate filaments.     

Two different correlations between nanoindentation modulus and mineral content in the bone-cartilage interface

The biomechanical properties of the zone of calcified cartilage (ZCC) in articulating joints are of clinical relevance due to the role ZCC plays in load transfer from cartilage to bone. To determine the micron-level mechanical properties and their correlation to mineral concentration in the ZCC, we combined nanoindentation (for micrometer level stiffness E(r) and hardness H) and quantitative back-scattered electron imaging or qBEI (for micrometer level mean calcium concentration Ca(Mean)) to study the ZCC-subchondral bone junction in 3 embedded human patellae. Nanoindentation line scans were correlated to qBEI analysis in the ZCC. The correlation between local stiffness and local mineral content was different in calcified cartilage compared to bone. The stiffness and hardness of calcified cartilage was typically lower than subchondral bone for the same mineral content. ZCC showed a wider range of variation in calcium content (1-28 wt %) compared to subchondral bone (16-26 wt %). 2D material property maps of the ZCC were generated from the mechanical-mineral correlation, showing that bands of high and low stiffness were found between the bone and tidemark, and between the ZCC and the unmineralized cartilage.     

Dissecting the protein-protein interface between beta-lactamase inhibitory protein and class A beta-lactamases

beta-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases at a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for inhibiting TEM-1 beta-lactamase and SME-1 beta-lactamase. Two hotspots of binding energy, one from each domain of BLIP, were identified (Zhang, Z., and Palzkill, T. (2003) J. Biol. Chem. 278, 45706-45712). This study has been extended to examine the amino acid sequence requirements of BLIP for binding to the SHV-1 beta-lactamase, which is a poor binding substrate (Ki= 1.1 microm), and the Bacillus anthracis Bla1 enzyme (Ki= 2.5 nm). The two hotspots previously identified as important for binding TEM-1 and SME-1 beta-lactamase were also found to be important for binding Bla1. The hotspot from the second domain of BLIP, however, does not make substantial contributions to SHV-1 binding. This may explain why BLIP binds to SHV-1 beta-lactamase with much weaker affinity than to the other three enzymes. Three regions, including two loops that insert into the active pocket of TEM-1 beta-lactamase and the Glu-73-Lys-74 buried charge motif, exhibit strikingly different effects on the binding affinity of BLIP toward the various enzymes when mutated and, therefore, act as specificity determinants. Analysis of double mutants of BLIP that combine specificity-determining residues suggests that these residues contribute to the poor affinity between the second domain of BLIP and SHV-1 beta-lactamase.     

Muscle strength and mineral densities in the mandible

Bone mineral density (BMD) in the femoral neck and lumbar spine was measured for 355 postmenopausal 48- to 56-year-old women and the BMD in five different regions in the mandible for 77. All 355 women were also classified according to the size of the masseter muscle. Both skeletal measures and the BMD of the buccal cortex distally from the foramen mentale were compared with the size of the masseter muscle. This study indicates that functional stress, caused by the masseter muscle, is involved in maintaining bone mineral density in edentulous regions of the mandible. Those individuals who are physically active or are bruxists may lose less mineral, after extractions of teeth, from those regions of the jaw bones where the muscles are attached.     

Characterization of the binding interface between the E-domain of Staphylococcal protein A and an antibody Fv-fragment

Staphylococcal protein A (SpA) is a cell-surface component of Staphylococcus aureus. In addition to the well-characterized interaction between SpA and the Fc-region of human IgG, an alternative binding interaction between SpA and the Fab-region of immunoglobulin domains encoded by the V(H)3 gene family has been described. To characterize structurally the interface formed by SpA repeats and type-3 V(H)-domains, we have studied the 32-kDa complex formed between an E-domain mutant (EZ4) and the Fv-fragment of the humanized anti-HER2 antibody (Hu4D5-8) using heteronuclear NMR spectroscopy. Protocols were established for efficient incorporation of (15)N, (13)C, and (2)H into EZ4 and the V(H)- and V(L)-domains of the Fv, allowing backbone resonances to be assigned sequentially for EZ4 and the V(H)-domain in both free and complexed states. Broadening of certain V(H)-resonances in the free and bound Fv-fragment suggests microsecond to millisecond time-scale motion in CDR3. Residues experiencing significant chemical shift changes of backbone (1)H(N), (15)N, and (13)CO resonances upon complex formation delineate contiguous surfaces on EZ4 and the V(H)-domain that define the binding interfaces of the two proteins. The interaction surfaces identified by chemical shift mapping are comprised of predominantly hydrophilic residues. This is in contrast to the SpA-Fc interface which is predominantly hydrophobic in nature. Further analysis of the surface properties suggests a probable binding orientation for SpA- and V(H)3-domains.     

A de novo designed protein protein interface

As an approach to both explore the physical/chemical parameters that drive molecular self-assembly and to generate novel protein oligomers, we have developed a procedure to generate protein dimers from monomeric proteins using computational protein docking and amino acid sequence design. A fast Fourier transform-based docking algorithm was used to generate a model for a dimeric version of the 56-amino-acid beta1 domain of streptococcal protein G. Computational amino acid sequence design of 24 residues at the dimer interface resulted in a heterodimer comprised of 12-fold and eightfold variants of the wild-type protein. The designed proteins were expressed, purified, and characterized using analytical ultracentrifugation and heteronuclear NMR techniques. Although the measured dissociation constant was modest ( approximately 300 microM), 2D-[(1)H,(15)N]-HSQC NMR spectra of one of the designed proteins in the absence and presence of its binding partner showed clear evidence of specific dimer formation.     

A hot-spot motif characterizes the interface between a designed ankyrin-repeat protein and its target ligand

Nonantibody scaffolds such as designed ankyrin repeat proteins (DARPins) can be rapidly engineered to detect diverse target proteins with high specificity and offer an attractive alternative to antibodies. Using molecular simulations, we predicted that the binding interface between DARPin off7 and its ligand (maltose binding protein; MBP) is characterized by a hot-spot motif in which binding energy is largely concentrated on a few amino acids. To experimentally test this prediction, we fused MBP to a transmembrane domain to properly orient the protein into a polymer-cushioned lipid bilayer, and characterized its interaction with off7 using force spectroscopy. Using this, to our knowledge, novel technique along with surface plasmon resonance, we validated the simulation predictions and characterized the effects of select mutations on the kinetics of the off7-MBP interaction. Our integrated approach offers scientific insights on how the engineered protein interacts with the target molecule.     

A structural criterion for the biological compatibility of materials

The principles of searching and selection of the polymeric materials compatible with biological tissues are offered, based on structural features of the hydration shells of biopolymers.     

The effects of royal jelly protein on bone mineral density and strength in ovariectomized female rats

[Purpose]Sex hormones deficiency leads to dramatically bone loss in particular postmenopausal women. Royal jelly has anti-osteoporosis effect due to maintain bone volume in that condition. We hypothesized that royal jelly protein (RJP, a latent residue after extracting royal jelly) also prevents bone deficient in ovariectomized (OVX) female rats, the animal model of postmenopausal women. [Methods]Female Sprague-Dawley rats (n = 30, 6 weeks age old) were sham operated (Sham; sham operated group, n = 7), OVX control group (OC, n = 7), OVX with low RJP intake group (ORL, n = 8), and OVX with high RJP intake group (ORH, n = 8) during 8 weeks experimental periods. In the end point of this experiment, the bone samples (lumbar spine, tibia, and femur) were surgically removed under anesthesia. These bone samples were evaluated bone mineral density (BMD) and bone strength.[Results]BMD of lumbar spine in RJP intake groups (ORL, ORH) were higher than that in OC group (p < 0.05 and p < 0.01) in RJP intake volume dependent manner. BMD of tibial proximal metaphysis and diaphysis in RJP intake groups were also higher than these in OC group (p < 0.01 and p < 0.01 / p < 0.05 and p < 0.001). In addition, breaking force of femur in RJP intake groups were significantly increase compared with that in OC group (p < 0.001 respectively). [Conclusion]These findings indicate that RJP contribute to prevent sex hormone related bone abnormality     

A trial to improve the analysis of boron in biological materials

For the spectrophotometric determination of boron in biological materials, two decomposition methods were used. One is the alkali fusion method, and the other is the acid decomposition method using a uniseal decomposition vessel. A long-range calibration curve for the former method was obtained by using an ultrapure sulfuric acid. The alkali fusion method was applied to samples into which paraboronophenylalanine (BPA) or its hydrochloric salt (BPA.HCl) had been injected. By the acid decomposition method using a uniseal vessel, 50 mg dried liver was digested by a mixed solution of 1 ml conc. sulfuric acid and 6 ml 30% hydrogen peroxide. A problem exists, however, in that the hydrogen peroxide remaining in the decomposition solution must be eliminated without loss of boron.