Multiple quantum filtered NMR studies of the interaction between collagen and water in the tendon

We studied the physical processes and the chemical reactions involved in magnetization transfer between water and large proteins, such as Collagen, in bovine Achilles tendon. Since the NMR spectrum for such proteins is broadened by very large dipolar interactions, the NMR peaks of the various functional groups on the protein cannot be separated from one another on the basis of their different chemical shifts. A further complication in observing the protein spectrum is the intense narrow peak of the abundant water. Thus, magnetization transfer (MT) within the protein or between water and the protein cannot rely on differences in the chemical shifts, as is commonly possible in liquids. We present a method that separates the protein spectrum from that of the water spectrum on the basis of their different intramolecular dipolar interactions, enabling exclusive excitation of either the protein or water. As a result, the protein spectrum as well as the effect of spin diffusion within the protein can be measured. In addition, the MT rates from the protein to water and vice versa can be measured. Two types of mechanisms were considered for the MT: chemical exchange- and dipolar interaction-related processes (such as NOE). They were distinguished by examining the effects of the following experimental conditions: (a) temperature; (b) pH; (c) ratio of D2O to H2O in the bathing liquid; (d) interaction of the protein with small molecules other than water, such as DMSO and methanol. Our results lead us to the conclusion that the MT is dominated below the freezing point by the dipolar interaction between the protein and water, while an exchange of protons between the protein and the water molecules is the most significant process above the freezing point. On the basis of the fact that the spin temperature is established for the protein on a time scale much shorter than that of the MT, we could measure protein spectra that are distinguished by the contributions made to them by the various functional groups; i.e., contributions of methylenes were distinguished from those of methyls.