This paper investigates an alternative explanation for widely reported paradoxical intracellular water properties. The most frequent biological explanation assumes water structure extending multiple layers from surfaces of compactly folded macromolecules to explain large amounts of perturbed water. Long range water structuring, however, contradicts molecular models widely accepted by the scientific majority. This study questions whether the paradoxical cell water could result from larger than expected amounts of first layer interfacial water on internal protein surfaces rather than structured multilayers. Native mammalian tendon is selected for the study because (1) the organ consists of highly compact structures of a single macromolecular protein--collagen, (2) molecular structure and geometry of collagen is well characterized by X-ray diffraction, (3) molecular structure extends to the macroscopic tendon level and (4) perturbed water behavior similar to cellular water is reported on tendon. Native tendon holds 1.6 g water/g dry mass. The 62% native water content simulates the water content of many cell types. MicroCT studies of tendon dilatometry as a function of hydration are measured and correlated to X-ray diffraction measurements of interaxial separation. Correlations show that native tendon has sufficient water for only a single monolayer of interfacial water. Thus the paradoxical properties of water in native tendon are first-layer interfacial water properties. Similar water behavior on globular proteins suggests that paradoxical cell water behavior could be caused by larger than expected amounts of first layer interfacial water on internal and external macromolecular surfaces of cell components.

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 h1 = 0.0658 g/g, (2) the Berendsen single water chain per cleft at h2 = 0.264 g/g, and (3) full monolayer coverage with six water chains per cleft level at h3 = 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.

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.