Anthony Nash

Preferential sites for intramolecular glucosepane cross-link formation in type I collagen: A thermodynamic study

The extracellular matrix (ECM) undergoes progressive age-related stiffening and loss of proteolytic digestibility due to an increase in concentration of advanced glycation end products (AGEs). The most abundant AGE, glucosepane, accumulates in collagen with concentrations over 100 times greater than all other AGEs. Detrimental collagen stiffening properties are believed to play a significant role in several age-related diseases such as osteoporosis and cardiovascular disease. Currently little is known of the potential location of covalently cross-linked glucosepane formation within collagen molecules; neither are there reports on how the respective cross-link sites affect the physical and biochemical properties of collagen. Using fully atomistic molecular dynamics simulations (MD) we have identified six sites where the formation of a covalent intra-molecular glucosepane cross-link within a single collagen molecule in a fibrillar environment is energetically favourable. Identification of these favourable sites enables us to align collagen cross-linking with experimentally observed changes to the ECM. For example, formation of glucosepane was found to be energetically favourable within close proximity of the Matrix Metalloproteinase-1 (MMP1) binding site, which could potentially disrupt collagen degradation.

Mapping intermolecular interactions and active site conformations: from human MMP-1 crystal structure to molecular dynamics free energy calculations

The zinc-dependent Matrix Metalloproteinases (MMPs) found within the extracellular matrix (ECM) of vertebrates are linked to pathological processes such as arthritis, skin ulceration and cancer. Although a general backbone proteolytic mechanism is understood, crystallographic data continue to suggest an active site that is too narrow to encompass the respective substrate. We present a fully parameterised Molecular Dynamics (MD) study of the structural properties of an MMP-1-collagen crystallographic structure (Protein Data Bank – 4AUO), followed by an exploration of the free energy surface of a collagen polypeptide chain entering the active site, using a combined meta-dynamics and umbrella sampling (MDUS) approach. We conclude that the interactions between MMP-1 and the collagen substrate are in good agreement with a number of experimental studies. As such, our unrestrained MD simulations and our MDUS results, which indicate an energetic barrier for a local uncoiling and insertion event, can inform future investigations of the collagen-peptide non-bonded association steps with the active site prior to proteolytic mechanisms. The elucidation of such free energy barriers provides a better understanding of the role of the enzyme in the ECM and is important in the design of future MMP inhibitors.

Computational study of glucosepane-water and hydrogen bond formation: an electron topology and orbital analysis.

The collagen protein provides tensile strength to the extracellular matrix in addition to localising cells, proteins and protein cofactors. Collagen is susceptible to a build up of glycation modifications as a result of an exceptionally long half-life. Glucosepane is a collagen cross-linking advanced glycation end product; the structural and mechanical effects of glucosepane are still the subjects of much debate. With the prospect of an ageing population, the management and treatment of age-related diseases is becoming a pressing concern. One area of interest is the isolation of hydrated glucosepane, which has yet to be reported at an atomistic level. This study presents a series of glucosepane–water complexes within an implicit aqueous environment. Electronic structure calculations were performed using density functional theory and a high level basis set. Hydrogen bonds between glucosepane and explicit water were identified by monitoring changes to covalent bonds, calculating levels of electron donation from Natural Bonding Orbital analysis and the detection of bond critical points. Hydrogen bond strength was calculated using second-order perturbation calculations. The combined results suggest that glucosepane is very hydrophilic, with the imidazole feature being energetically more attractive to water than either hydroxyl group, although all hydrogen bonds, regardless of bond strength, were electrostatic in nature. Our results are in growing support of an earlier hypothesis that cross-links may result in an increase in interstitial water retention, which would permit the collagen fibril to swell, thereby potentially affecting the tensile and compression properties and biological function of connective tissues.

Combining nano-physical and computational investigations to understand the nature of “aging” in dermal collagen

The extracellular matrix of the dermis is a complex, dynamic system with the various dermal components undergoing individual physiologic changes as we age. Age-related changes in the physical properties of collagen were investigated in particular by measuring the effect of aging, most likely due to the accumulation of advanced glycation end product (AGE) cross-links, on the nanomechanical properties of the collagen fibril using atomic force microscope nano-indentation. An age-related decrease in the Young’s modulus of the transverse fibril was observed (from 8.11 to 4.19 GPa in young to old volunteers, respectively, P<0.001). It is proposed that this is due to a change in the fibril density caused by age-related differences in water retention within the fibrils. The new collagen–water interaction mechanism was verified by electronic structure calculations, showing it to be energetically feasible.

Intra-molecular lysine-arginine derived advanced glycation end-product cross-linking in Type I collagen: A molecular dynamics simulation study.

Covalently cross-linked advanced glycation end products (AGE) are among the major post-translational modifications to proteins as a result of non-enzymatic glycation. The formation of AGEs has been shown to have adverse effects on the properties of the collagenous tissue; they are even linked to a number of age related disorders. Little is known about the sites at which these AGEs form or why certain sites within the collagen are energetically more favourable than others. In this study we have used a proven fully atomistic molecular dynamics approach to identify six sites where the formation of the intra-molecular 3-deoxyglucosone-derived imidazolium cross-link (DOGDIC) is energetically favourable. We have also conducted a comparison of these positions with those of the more abundant glucosepane cross-link, to determine any site preference. We show that when we consider both lysine and arginine AGEs, they exhibit a prevalence to form within the gap region of the collagen fibril.

ForceGen: atomic covalent bond value derivation for Gromacs

A large number of crystallographic protein structures include ligands, small molecules and post-translational modifications. Atomic bond force values for computational atomistic models of post-translational or non-standard amino acids, metal binding active sites, small molecules and drug molecules are not readily available in most simulation software packages. We present ForceGen, a Java tool that extracts the bond stretch and bond angle force values and equilibrium values from the Hessian of a Gaussian vibrational frequency analysis. The parameters are compatible with force fields derived using the second order tensor of the Hessian. The output is formatted with the Gromacs topology in mind. This study further demonstrates the use of ForceGen over the quantum mechanically derived structures of a small organic solvent, a naturally occurring protein crosslink derived from two amino acids following post-translational modification and the amino acid ligands of a zinc ion. We then derive Laplacian bond orders to understand how the resulting force values relate back to the quantum mechanical model. The parameterisation of the organic solvent, toluene, was verified using Molecular Mechanics simulations. The structural data from the simulation compared well with the quantum mechanical structure and the system density compared well with experimental values.

Relative orientation of collagen molecules within a fibril: a homology model for homo sapiens type I collagen

Type I collagen is an essential extracellular protein that plays an important structural role in tissues that require high tensile strength. However, owing to the molecule’s size, to date no experimental structural data are available for the Homo sapiens species. Therefore, there is a real need to develop a reliable homology model and a method to study the packing of the collagen molecules within the fibril. Through the use of the homology model and implementation of a novel simulation technique, we have ascertained the orientations of the collagen molecules within a fibril, which is currently below the resolution limit of experimental techniques. The longitudinal orientation of collagen molecules within a fibril has a significant effect on the mechanical and biological properties of the fibril, owing to the different amino acid side chains available at the interface between the molecules.

Effect on the mechanical properties of type I collagen of intra-molecular lysine-arginine derived advanced glycation end-product cross-linking

Non-enzymatic advanced glycation end product (AGE) cross-linking of collagen molecules has been hypothesised to result in significant changes to the mechanical properties of the connective tissues within the body, potentially resulting in a number of age related diseases. We have investigated the effect of two of these cross-links, glucosepane and DOGDIC, on the tensile and lateral moduli of the collagen molecule through the use of a steered molecular dynamics approach, using previously identified preferential formation sites for intra-molecular cross-links. Our results show that the presence of intra-molecular AGE cross-links increases the tensile and lateral Young’s moduli in the low strain domain by between 3.0–8.5% and 2.9–60.3% respectively, with little effect exhibited at higher strains.

Headache and type 2 diabetes association: a US national ambulatory case-control study.

Objective: We investigate the joint observation between type 2 diabetes and headache using a cross-sectional case-control study of a US ambulatory dataset. Background: Recent whole-population cohort studies propose that type 2 diabetes may have a protective effect against headache prevalence. With headaches ranked as a leading cause of disability, headache-associated comorbidities could help identify shared molecular mechanisms. Methods: We performed a cross-sectional case-control study using the US National Ambulatory Medical Care Survey, 2009, on the joint observation between headache and specific comorbidities, namely type 2 diabetes, hypertension and anxiety, for all patients between 18 and 65 years of age. The odds ratio of having a headache and a comorbidity were calculated using conditional logistic regression, controlling for gender and age over a study population of 3,327,947 electronic health records in the absence of prescription medication data. Results: We observed estimated odds ratio of 0.89 (95% CI: 0.83-0.95) of having a headache and a record of type 2 diabetes over the population, and 0.83 (95% CI: 2.02-2.57) and 0.89 (95% CI: 3.00-3.49) for male and female, respectively. Conclusions: We find that patients with type 2 diabetes are less likely to present a recorded headache indication. Patients with hypertension are almost twice as likely of having a headache indication and patients with an anxiety disorder are almost three times as likely. Given the possibility of confounding indications and prescribed medications, additional studies are recommended.

Modelling water diffusion in plasticizers: development and optimization of a force field for 2,4-dinitroethylbenzene and 2,4,6-trinitroethylbenzene

A classical all-atom force field has been developed for 2,4,6-trinitroethylbenzene and 2,4-dinitroethylbenzene and applied in molecular dynamics simulations of the two pure and two mixed plasticizer systems. Bonding parameters and partial charges were derived through electronic and geometry optimization of the single molecules. The other required parameters were derived from values already available in the literature for generic nitro aromatic compounds, which were adjusted to reproduce to a high level of accuracy the densities of 2,4-dinitroethylbenzene, 2,4,6-trinitroethylbenzene and the energetic plasticizers K10 and R8002. This force field has been applied to both K10 and R8002, which when used as plasticizers form an energetic binder with nitrocellulose. Nitrocellulose decomposes in storage, under varying conditions, but in particular where it may become increasingly dry. Following the derivation of the force field, we have therefore applied it to calculate water diffusion coefficients for each of the different materials at 298 K and 338 K, thereby providing a starting point for understanding water behaviour in a nitrocellulose binder.