Insights into anti-D formation in carriers of RhD variants through studies of 3D intraprotein interactions.

BACKGROUND: Many RhD variants associated with anti-D formation (partial D) in carriers exposed to the conventional D antigen carry mutations affecting extracellular loop residues. Surprisingly, some carry mutations affecting transmembrane or intracellular domains, positions not thought likely to have a major impact on D epitopes. STUDY DESIGN AND METHODS: A wild-type Rh trimer (RhD1 RhAG2 ) was modeled by comparative modeling with the human RhCG structure. Taking trimer conformation, residue accessibility, and position relative to the lipid bilayer into account, we redefine the domains of the RhD protein. We generated models for RhD variants carrying one or two amino acid substitutions associated with anti-D formation in published articles (25 variants) or abstracts (12 variants) and for RHD*weak D type 38. We determined the extracellular substitutions and compared the interactions of the variants with those of the standard RhD. RESULTS: The findings of the three-dimensional (3D) analysis were correlated with anti-D formation for 76% of RhD variants: 15 substitutions associated with anti-D formation concerned extracellular residues, and structural differences in intraprotein interactions relative to standard RhD were observed in the others. We discuss the mechanisms by which D epitopes may be modified in variants in which the extracellular residues are identical to those of standard RhD and provide arguments for the benignity of p.T379M (RHD*DAU0) and p.G278D (RHD*weak D type 38) in transfusion medicine. CONCLUSION: The study of RhD intraprotein interactions and the precise redefinition of residue accessibility provide insight into the mechanisms through which RhD point mutations may lead to anti-D formation in carriers.

Numerical modeling of the effects hydration and number of hydrogen bonds on the mechanical properties of the tropocollagen molecule.

Bone aging involves structural and molecular modifications, especially at the level of type I tropocollagen. This macromolecule shows two main age-related alterations, which are the decrease of both molecular diameter (due to the loss of hydration) and number of hydrogen bonds. In this work, it is proposed to investigate the influence of these two parameters (molecular diameter and number of hydrogen bonds) on the mechanical behavior of tropocollagen using finite element method. To this end, a novel three-dimensional finite element model of collagen molecule accounting for hydrogen bonds was developed. Then, a numerical design of experiments for the diameter of tropocollagen and variations in the number of hydrogen bonds has been established. The mechanical properties ("load-strain" curve and apparent Young's modulus) of the collagen molecule were obtained by employing the proposed model to uniaxial tensile tests. The parametric study demonstrates that the mechanical properties of tropocollagen are slightly affected by the rate of hydration but considerably affected by variation of the number of hydrogen bonds. Finally, a fitted analytical function was deduced from the above results showing effects of the two parameters (hydration rate and hydrogen bonds) on the apparent Young's modulus of tropocollagen. This study could be useful to understand the influence of structural age modifications of tropocollagen on the macroscopic mechanical properties of bone.

Effect of collagenase-gelatinase ratio on the mechanical properties of a collagen fibril: a combined Monte Carlo-molecular dynamics study.

Loading in cartilage is supported primarily by fibrillar collagen, and damage will impair the function of the tissue, leading to pathologies such as osteoarthritis. Damage is initiated by two types of matrix metalloproteinases, collagenase and gelatinase, that cleave and denature the collagen fibrils in the tissue. Experimental and modeling studies have revealed insights into the individual contributions of these two types of MMPs, as well as the mechanical response of intact fibrils and fibrils that have experienced random surface degradation. However, no research has comprehensively examined the combined influences of collagenases and gelatinases on collagen degradation nor studied the mechanical consequences of biological degradation of collagen fibrils. Such preclinical examinations are required to gain insights into understanding, treating, and preventing degradation-related cartilage pathology. To develop these insights, we use sequential Monte Carlo and molecular dynamics simulations to probe the effect of enzymatic degradation on the structure and mechanics of a single collagen fibril. We find that the mechanical response depends on the ratio of collagenase to gelatinase-not just the amount of lost fibril mass-and we provide a possible mechanism underlying this phenomenon. Overall, by characterizing the combined influences of collagenases and gelatinases on fibril degradation and mechanics at the preclinical research stage, we gain insights that may facilitate the development of targeted interventions to prevent the damage and loss of mechanical integrity that can lead to cartilage pathology.

The Role of Matrix Composition in the Mechanical Behavior of Bone.

PURPOSE OF REVIEW: While thinning of the cortices or trabeculae weakens bone, age-related changes in matrix composition also lower fracture resistance. This review summarizes how the organic matrix, mineral phase, and water compartments influence the mechanical behavior of bone, thereby identifying characteristics important to fracture risk. RECENT FINDINGS: In the synthesis of the organic matrix, tropocollagen experiences various post-translational modifications that facilitate a highly organized fibril of collagen I with a preferred orientation giving bone extensibility and several toughening mechanisms. Being a ceramic, mineral is brittle but increases the strength of bone as its content within the organic matrix increases. With time, hydroxyapatite-like crystals experience carbonate substitutions, the consequence of which remains to be understood. Water participates in hydrogen bonding with organic matrix and in electrostatic attractions with mineral phase, thereby providing stability to collagen-mineral interface and ductility to bone. Clinical tools sensitive to age- and disease-related changes in matrix composition that the affect mechanical behavior of bone could potentially improve fracture risk assessment.

Nanomechanical mapping of hydrated rat tail tendon collagen I fibrils.

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 µm in length, were acquired at indentation speeds around 10(5) nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10(5) nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.

Demonstrating collagen tendon fibril segments involvement in intrinsic tendon repair.

Severed tendons can undergo regenerative healing, intrinsic tendon repair. Fibrillogenesis of chick tendon involves "collagen fibril segments" (CFS), which are the building blocks of collagen fibers that make up tendon fascicles. The CFS are 10.5 micron in length, composed of tropocollagen monomers arranged in parallel arrays. Rather than incorporating single tropocollagen molecules into growing collagen fibers, incorporating large CFS units is the mechanism for generating collagen fibers. Is intrinsic tendon repair through the reestablishment of tendon embryogenesis? Gentamicin treated 10-day-old chick embryo tendons released CFS were fluorescently tagged with Rhodamine (Rh). Organ cultured severed 14-day-old embryo tendon explants received Rh tagged CFS. At day 4 auto fluorescent tagged CFS were identified at the severed tendon ends by fluorescent microscopy. Accumulation of fluorescent tagged CFS was exclusively localized to the severed ends of tendon explants. Parallels between collagen fiber growth during embryonic fibrillogenesis and tendon repair reveal CFS incorporation is responsible for collagen fibers growth. CFS incorporation into frayed collagen fibers from severed tendons is the proposed mechanism for intrinsic tendon repair, which is an example of regenerative repair.

Molecular and mesoscale mechanisms of osteogenesis imperfecta disease in collagen fibrils.

Osteogenesis imperfecta (OI) is a genetic disorder in collagen characterized by mechanically weakened tendon, fragile bones, skeletal deformities, and in severe cases, prenatal death. Although many studies have attempted to associate specific mutation types with phenotypic severity, the molecular and mesoscale mechanisms by which a single point mutation influences the mechanical behavior of tissues at multiple length scales remain unknown. We show by a hierarchy of full atomistic and mesoscale simulation that OI mutations severely compromise the mechanical properties of collagenous tissues at multiple scales, from single molecules to collagen fibrils. Mutations that lead to the most severe OI phenotype correlate with the strongest effects, leading to weakened intermolecular adhesion, increased intermolecular spacing, reduced stiffness, as well as a reduced failure strength of collagen fibrils. We find that these molecular-level changes lead to an alteration of the stress distribution in mutated collagen fibrils, causing the formation of stress concentrations that induce material failure via intermolecular slip. We believe that our findings provide insight into the microscopic mechanisms of this disease and lead to explanations of characteristic OI tissue features such as reduced mechanical strength and a lower cross-link density. Our study explains how single point mutations can control the breakdown of tissue at much larger length scales, a question of great relevance for a broad class of genetic diseases.

Deformation rate controls elasticity and unfolding pathway of single tropocollagen molecules.

Collagen is an important structural protein in vertebrates and is responsible for the integrity of many tissues like bone, teeth, cartilage and tendon. The mechanical properties of these tissues are primarily determined by their hierarchical arrangement and the role of the collagen matrix in their structures. Here we report a series of Steered Molecular Dynamics (SMD) simulations in explicit solvent, used to elucidate the influence of the pulling rate on the Young's modulus of individual tropocollagen molecules. We stretch a collagen peptide model sequence [(Gly-Pro-Hyp)(10)](3) with pulling rates ranging from 0.01 to 100 m/s, reaching much smaller deformation rates than reported in earlier SMD studies. Our results clearly demonstrate a strong influence of the loading velocity on the observed mechanical properties. Most notably, we find that Young's modulus converges to a constant value of approximately 4 GPa tangent modulus at 8% tensile strain when the initially crimped molecule is straightened out, for pulling rates below 0.5 m/s. This enables us for the first time to predict the elastic properties of a single tropocollagen molecule at physiologically and experimentally relevant pulling rates, directly from atomistic-level calculations. At deformation rates larger than 0.5 m/s, Young's modulus increases continuously and approaches values in excess of 15 GPa for deformation rates larger than 100 m/s. The analyses of the molecular deformation mechanisms show that the tropocollagen molecule unfolds in distinctly different ways, depending on the loading rate, which explains the observation of different values of Young's modulus at different loading rates. For low pulling rates, the triple helix first uncoils completely at 10%-20% strain, then undergoes some recoiling in the opposite direction, and finally straightens for strains larger than 30%. At intermediate rates, the molecule uncoils linearly with increasing strain up to 35% strain. Finally, at higher velocities the triple helix does not uncoil during stretching.

Single molecule effects of osteogenesis imperfecta mutations in tropocollagen protein domains.

Osteogenesis imperfecta (OI) is a genetic disease characterized by fragile bones, skeletal deformities and, in severe cases, prenatal death that affects more than 1 in 10,000 individuals. Here we show by full atomistic simulation in explicit solvent that OI mutations have a significant influence on the mechanical properties of single tropocollagen molecules, and that the severity of different forms of OI is directly correlated with the reduction of the mechanical stiffness of individual tropocollagen molecules. The reduction of molecular stiffness provides insight into the molecular-scale mechanisms of the disease. The analysis of the molecular mechanisms reveals that physical parameters of side-chain volume and hydropathy index of the mutated residue control the loss of mechanical stiffness of individual tropocollagen molecules. We propose a model that enables us to predict the loss of stiffness based on these physical characteristics of mutations. This finding provides an atomistic-level mechanistic understanding of the role of OI mutations in defining the properties of the basic protein constituents, which could eventually lead to new strategies for diagnosis and treatment the disease. The focus on material properties and their role in genetic diseases is an important, yet so far only little explored, aspect in studying the mechanisms that lead to pathological conditions. The consideration of how material properties change in diseases could lead to a new paradigm that may expand beyond the focus on biochemical readings alone and include a characterization of material properties in diagnosis and treatment, an effort referred to as materiomics.

New approaches for study of mechanisms of bone demineralization due to microgravity.

NASA: Changes in nano-scale subsystems of rat femurs due to the axial unloading of hindlimbs are studied by means of electron paramagnetic resonance (EPR). After irradiation by 60Co isotopes, the results indicate that weightlessness simulation leads to formation of free radicals in tropocollagen molecules and to a reduction in the amount of CO2 radicals, located on the surface of bioapatite nanocrystals.^ieng