Andres Rahal

Collagen structure: the molecular source of the tendon magic angle effect.

This review of tendon/collagen structure shows that the orientational variation in MRI signals from tendon, which is referred to as the “magic angle” (MA) effect, is caused by irreducible separation of charges on the main chain of the collagen molecule. These charges are held apart in a vacuum by stereotactic restriction of protein folding due in large part to a high concentration of hydroxyproline ring residues in the amino acids of mammalian collagen. The elevated protein electrostatic energy is reduced in water by the large dielectric constant of the highly polar solvent (κ ∼ 80). The water molecules serve as dielectric molecules that are bound by an energy that is nearly equivalent to the electrostatic energy between the neighboring positive and negative charge pairs in a vacuum. These highly immobilized water molecules and secondary molecules in the hydrogen‐bonded water network are confined to the transverse plane of the tendon. Orientational restriction causes residual dipole coupling, which is directly responsible for the frequency and phase shifts observed in orientational MRI (OMRI) described by the MA effect. Reference to a wide range of biophysical measurements shows that native hydration is a monolayer on collagen hm = 1.6 g/g, which divides into two components consisting of primary hydration on polar surfaces hpp = 0.8 g/g and secondary hydration hs = 0.8 g/g bridging over hydrophobic surface regions. Primary hydration further divides into side‐chain hydration hpsc = 0.54 g/g and main‐chain hydration hpmc = 0.263 g/g. The main‐chain fraction consists of water that bridges between charges on the main chain and is responsible for almost all of the enthalpy of melting ΔH = 70 J/g‐dry mass. Main‐chain water bridges consist of one extremely immobilized Ramachandran water bridge per tripeptide hRa = 0.0658 g/g and one double water bridge per tripeptide hdwb = 0.1974 g/g, with three water molecules that are sufficiently slowed to act as the spin‐lattice relaxation sink for the entire tendon. J. Magn. Reson. Imaging 2007.

An NMR method to characterize multiple water compartments on mammalian collagen

A molecular model is proposed to explain water 1H NMR spin‐lattice relaxation at different levels of hydration (NMR titration method) on collagen. A fast proton exchange model is used to identify and characterize protein hydration compartments at three distinct Gibbs free energy levels. The NMR titration method reveals a spectrum of water motions with three well‐separated peaks in addition to bulk water that can be uniquely characterized by sequential dehydration. Categorical changes in water motion occur at critical hydration levels h (g water/g collagen) defined by integral multiples N = 1, 4 and 24 times the fundamental hydration value of one water bridge per every three amino acid residues as originally proposed by Ramachandran in 1968. Changes occur at (1) the Ramachandran single water bridge between a positive amide and negative carbonyl group at h 1 = 0.0658 g/g, (2) the Berendsen single water chain per cleft at h 2 = 0.264 g/g, and (3) full monolayer coverage with six water chains per cleft level at h 3 = 1.584 g/g. The NMR titration method is verified by comparison of measured NMR relaxation compartments with molecular hydration compartments predicted from models of collagen structure. NMR titration studies of globular proteins using the hydration model may provide unique insight into the critical contributions of hydration to protein folding.

Imaging of the Diabetic Foot Diagnostic Dilemmas

Multiple diagnostic imaging modalities are available and beneficial for the evaluation of the diabetic foot. There is not yet “one best test” for sorting out the diagnostic dilemmas commonly encountered. The differentiation of cellulitis alone from underlying osteomyelitis and the early detection of abscesses remain important diagnostic goals. Equally important, differentiation of osteomyelitis and neuroarthropathy remains a difficult job. This is often compounded by postoperative diabetic foot states status after reconstruction. Diagnostic evaluation often involves multiple studies that are complementary and that include conventional radiography, computed tomography, nuclear medicine scintigraphy, magnetic resonance imaging, ultrasonography, and positron emission tomography.

Micro-CT dilatometry measures of molecular collagen hydration using bovine extensor tendon.

PURPOSE This article introduces a new method to study macromolecular hydration using micro-CT dilatometry. The complexity of hydration dependence on solvent temperature, pH, ionic charge, ionic activity, and ionic radii are barriers to comprehensive understanding of protein function. The crystalline character of collagen-tendon suggests that tendon dilatometry may give direct access to measures of molecular tropocollagen solvation response. METHODS The molecular basis of the stoichiometric hydration model (SHM) provides tools to validate bovine tendon as a model to study protein-solvent shape response by micro-CT measures of tendon diameter, length, and mass during dehydration. The SHM relates macroscopic properties to molecular properties of water interacting with the surface of collagen molecules. There are marked changes at critical SHM hydration levels h = 0.0653, 0.262, and 0.724 g water/g dry weight. RESULTS Micro-CT analysis of the length, diameter, and volume combined with gravimetric measures of tendon mass as a function of hydration h (g water/g dry solid) shows asymmetric changes in length, diameter, and density as predicted by SHM. The collagen molecules perturb water properties of polar hydration N=11 waters per tripeptide unit or h approximately 0.724 g/g to confirm MDS prediction of elevated hydration density 20%-50% higher than bulk water. CONCLUSIONS Results validate the use of tendon dilatometry amplification factors of 10(6)-10(8) as an effective model to investigate protein molecule shape change response to solvent molecules. The tendon model for the first time allows direct study of protein hydration and functional response under physiological conditions.

SU‐GG‐I‐128: Biophysical Studies of Tendon to Elucidate Magic Angle MRI

Purpose: The “Magic Angle Effect” observed in cartilage, tendon and other collagen rich tissues of the extracellular matrix have largely unused potential for evaluation of musculo‐skeletal disease processes. These studies seek to improve understanding of the molecular basis for magic angle phenomena based on collagen hydration. We hypothesize that improved understanding will provide a conceptual framework and extend the ability of musculo‐skeletal radiologists to design protocols using MRI orientational contrast for better accuracy and specificity. Method and Materials: These studies use Differential Scanning Calorimetry(DSC) to directly measure the enthalpy and entropy of water bound to rat tail collagen/tendon at different hydration levels. Water vapor sorption and recovery rehydration rates of rat tail collagen/tendon at 22 °C were also measured. Measured bound water fractions are compared to the theoretical values (h=0.263 g/g) predicted from the molecular structure of collagen. Bound water hydration measured with both methods agreed with the molecular prediction of water bound to the protein back bone. Statistical analyses performed using Graphpad Prism. Results: The water vapor sorption and DSC studies of collagen/tendon at 22 °C show that both equilibrium hydration and enthalpy are linear functions of relative humidity up to critical hydration of the protein backbone h = 0.26 g/g. The water bridge hydration hypothesis identifies three hydration water fractions in direct contact with the protein that differ in motional/orientational properties from bulk water. Comparison to T1 and T2 relaxation rate and orientational studies shows the relaxation rate of tendon is determined by fast exchange of water between these motional and orientational restricted water fractions. Conclusion: This work offers evidence for the important role of protein main chain hydration in determining MRI contrast due to dielectric binding of polar water molecules interacting with partial electric charges separated on the backbone by steric restrictions of the collagen molecule.