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Cell Biology International (2006) 30, 6673 (Printed in Great Britain)
An NMR method to characterize multiple water compartments on mammalian collagen
Gary D. Fullertona*, Elena Nesa, Maxwell Amuraoa, Andres Rahala, Lada Krasnosselskaiaa and Ivan Cameronb
aRadiology Department, University of Texas HSCSA, Floyd Curl Drive, San Antonio, TX 78229-3900, United States
bCellular and Structural Biology Department, University of Texas HSCSA, Floyd Curl Drive, San Antonio, TX 78229-3900, United States
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
Keywords: Collagen, Protein hydration, NMR relaxometry, MRI, Proton relaxation, Tendon, Hydration compartments.
The scientific community generally accepts that protein hydration and protein induced water structuring are fundamental sources of protein structural behaviors that support life functions. Collagen is frequently selected as the model protein to elucidate structural and hydration relationships (Leikin et al., 1994, 1995, 1997, 2002; Privalov, 1982). Tendon has high concentration of Type I collagen approaching 100% of the dry biomass in some instances. Molecular tropocollagen crystallizes spontaneously and as a result significant new structural information is available with ever improving accuracy from X-ray diffraction studies of both native and molecular analogues of collagen (Bella and Berman, 1996; Bella et al., 1994, 1995, 1996; Berisio et al., 2002; Fraser et al., 1979; Kramer et al., 1998, 1999, 2000, 2001; Miller and Scheraga, 1976; Okuyama et al., 1977; Ramachandran, 1967; Rich and Crick, 1961; Yonath and Traub, 1969). The more recent, high-resolution studies consistently show a water bridge network surrounding the collagen molecule.
Structural studies provide a molecular model of collagen supporting the existence of three distinct categories of water bridges. The most tightly bound consists of one highly immobilized “water bridge” per every three protein residues h
This study tests the hypothesis that that the NMR spin-lattice relaxation time of water on native collagen is not characteristic of any of the three molecular compartments but a weighted average due to fast exchange between three hydration fractions or water phases on the protein surface (Zimmerman and Brittin, 1957). The relaxation characteristics of all three water phases can, however, be extracted and characterized by sequential NMR measurements using a titration of water content (Fullerton et al., 1982, 1986). Tendon is uniquely suited to identifying the molecular origin of the three hydration fractions that may be typical of proteins in general. The collagen molecule is highly immobilized and aligned in protein fiber structure that suppresses proton NMR signal from the protein due to static dipole coupling. The study confirms that proton NMR signal emanates only from the liquid water that shows the presence of three distinct hydration fractions in fast exchange equilibrium. The compartments extracted using the NMR titration method quantitatively confirm the three molecular water bridge environments identified in the preceding paragraph. The correlation of protein NMR titration measurements with a molecular model of water bridges holds promise as a general model to evaluate hydration of both fibrillar and globular proteins.
2 Materials and methods
2.1 NMR titration measurements
Samples of bovine flexor tendon from three animals (age and sex unknown) were obtained from a local slaughter house, dissected on site, diced to 3
Each relaxation measurement consisted of 31 amplitude measurements with a total of 30 sequential delay times with even increments of &007E;20
The original sample weights were approximately 5.4
2.2 NMR titration analysis of spin-lattice relaxation data
The water bridge hydration hypothesis identifies three hydration water fractions in addition to a bulk water phase as shown in Fig. 2. For the protein in dilute solution the hydration fractions are assumed to be in fast exchange equilibrium on T Fig. 2 (a) Conceptual cartoon for Zone 1 includes bulk water and shows the Gibbs free energy relationships of water molecules in different water bridge bonding arrangements on the collagen molecule. At low hydration levels water molecules accumulate in the “Water Bridges” at the lowest free energy level. Higher energy compartments are filled at higher hydrations by sequential filling of the “Cleft Water” and “Interfacial Monolayer Water until water spills over into the “Bulk” water compartment. (b) There is fast exchange of protons between these compartments when the tissue contains sufficient water to fill all the hydration compartments and maintains a bulk water fraction as well. As protons sample all compartments during decay the NMR spin-lattice decay is monoexponential.
(a) Conceptual cartoon for Zone 1 includes bulk water and shows the Gibbs free energy relationships of water molecules in different water bridge bonding arrangements on the collagen molecule. At low hydration levels water molecules accumulate in the “Water Bridges” at the lowest free energy level. Higher energy compartments are filled at higher hydrations by sequential filling of the “Cleft Water” and “Interfacial Monolayer Water until water spills over into the “Bulk” water compartment. (b) There is fast exchange of protons between these compartments when the tissue contains sufficient water to fill all the hydration compartments and maintains a bulk water fraction as well. As protons sample all compartments during decay the NMR spin-lattice decay is monoexponential.
Zone 2 (linear): bulk water depleted leaving cleft water and water bridge compartments full and in fast exchange with a variable interface monolayer compartment;
Zone 3 (linear): bulk water and interface monolayer compartments depleted, water bridge compartment full and in fast exchange with a variable cleft water compartment; and
Zone 4 (non-linear): bulk, interface monolayer and cleft water compartments depleted leaving tripeptide segments on the protein either occupied or not occupied (with or without water bridges). Each tropocollagen has many water bridge sites (&007E;1000) to yield mean relaxation rate by spin-diffusion during transition from full bridges to completely dry protein relaxation rates.
The dilute protein case is shown in Figs. 2 and 3 as Zone 1 where 1/h
“Zonal Analysis” of relaxation rate versus the reciprocal of hydration 1/h. A multi-segment non-linear least squares regression fit (R2
The monolayer hydration h
Zone 4 differs from the other zones because it describes the transition from collagen with water bridges to collagen without bridges. There is a broad spectrum of NMR signal from protons (hydrogen nuclei) covalently bound in the protein. A fraction of this protein signal appears in the water resonance range. In Zone 4 the proton signal comes from solid-like protein in two possible states either with or without water bridges. The observed change in relaxation rate indicates that protein with water relaxes slightly more rapidly than collagen without water bridges. The water bridges accelerate relaxation for protein without structural water. This is likely through the well known mechanism of spin-diffusion in solids (Edzes and Samulski, 1977). Thus there are two protein categories: category A with water bridges and category B without water. As the signal from “solid like” water is much smaller than from protein we can neglect the signal amplitude due to the water and calculate the relaxation rate as follows: R R f f h h f
This last equation R
Multi-segment or zonal fast exchange equations for spin-lattice relaxation for water on collagen
2.3 Statistical analysis
The results from samples S1 are shown in Fig. 3. Results on S2 were similar but not shown for brevity.
The details of the least squares fits are summarized in Table 2 for both the tendon S1 and S2 measurement series. The goodness of fit for tendon S1 was R2
Fitting constants for statistical fits to NMR titration studies
Correlation times for water calculated from data in Table 2 using BPP theory (Bloembergen et al., 1948)
4.1 NMR titration method
Comparison of the NMR titration measurements shown in Fig. 3 (data points) with predictions of the four zone multi-component molecular model of collagen hydration (solid line) calculated with parameters from Table 1 demonstrates the capacity of fast exchange relationships to accurately describe the relaxation rate of water on collagen. The model predictions of sharp demarcations at h
The Ramachandran proposal of one direct hydrogen bond per tripeptide, one water bridge and a second non-charge bridge water molecule was necessary to complete the two-bond model proposed by his group. (a) Shows configuration when peptide three is other than hydroxyproline. (b) Shows the alternative tripeptide hydrogen bonding configuration when hydroxyproline occupies position three in Chain A. In both instances h
4.2 Water bridges
As shown in Fig. 5 the function of the water bridge and direct hydrogen bond is to serve as mechanisms to reduce the electrostatic energy of the protein by transferring positive charge from amide sites to compensate negative charge from carbonyl groups. Direct hydrogen bonds occur when the separation is less than 3
Molecular water serves as a dielectric and thereby reduces the in vacuo electrostatic energy of the collagen molecule by aligning with the electric field generated between positive amide and negative carbonyl charge sites on the neighboring α-protein chains. Water bridges are highly immobilized such that the relaxation rates of protein with water bridge R
4.3 Cleft water
We define cleft water as the additional three water molecules per tripeptide that bind in a hydrogen bonded network to the protein as well as to the structural water bridge (also located in the cleft) to complete the four-water molecule chain per every three peptide residues in the molecular grooves of the collagen triple helix proposed as by Berendsen (1962) and Fullerton and Amurao (2005). We reason on the basis of geometry that cleft waters participate in double water bridges between the remaining main chain amide group and neighboring carbonyl groups; recall one of the three amides participates in a direct bond (Rich and Crick, 1955), a second in a single water bridge (Ramachandran and Chandrasekharan, 1968) and only one remains. As discussed by Westerhof (1993) a single water binds to the positive amide but branches into double water bridges to bind with three water molecules to two negative carbonyl groups when separations are too great for single water bridges. Thus direct bonds (shortest separation), structural waters (intermediate separation) and cleft water (greatest separation) use four waters to fill all the available hydrogen bonding sites on the collagen protein main chain. All remaining water bridges originate from less confined side chains such as the positive hydroxyl group on hydroxyproline.
As shown in Table 2 the R
4.4 Interfacial monolayer water
As seen in Fig. 3 and Tables 2 and 3 the relaxation times of the outer most water compartment on the two native tendon samples measured in Zone 2 do not differ significantly. However, the mean R
The maximum extent of interfacial monolayer water is difficult to determine accurately from the measurements in Fig. 3. Prior experiments on other protein solutions (Hallenga and Koenig, 1976) show that under dilute conditions of Zone 1 the straight line extrapolates to the relaxation rate measured for bulk water or R
4.5 Protein relaxation
The protein relaxation rates for S1 and S2 are significantly different even though the relaxations of the water fractions are to all purposes identical. This difference will require further investigation but is likely due to difference in collagen crosslinking due to life-time stress and other animal specific biomolecular factors.
4.6 Application of NMR titration to lysozyme
The NMR titration method was developed originally as a phenomenological method without theoretical basis in an attempt to understand and predict MR image contrast. Lysozyme was studied as model globular protein system (Fullerton et al., 1986). The re-evaluation of the published results allows testing of the molecular hydration model. The molecular weight of lysozyme is A
The NMR titration method provides measurements to directly relate water NMR relaxation to an underlying molecular model of collagen hydration. Although the description of hydration fractions as integer multiples of the Ramachandran hydration fraction may be merely fortuitous, the regularity of protein's primary and secondary structure provides a geometric foundation consistent with such a stoichiometric hydration relationship. In addition the hydration model encompasses a wide range of difficult to explain empirical observations of protein hydration effects that have eluded coherent molecular explanation for many years.
The NMR spin-lattice relaxation “sink” for collagen is identified as three water molecules per every three peptide forming double water bridges between a single positive amide group and two carbonyl groups on the protein main chain. Comparison with results on lysozyme implies similar intimate association of three water molecules with the main chain of globular proteins in both α-helix and β-pleat configurations. Such a regular stoichiometric association of internal water with native, folded proteins is, however, contrary to the majority view of the scientific community and should be viewed with caution.
The NMR titration method thus allows experimental confirmation of hydration model calculations and predictions of hydration properties for lysozyme and possibly other proteins directly from molecular composition. Collagen hydration rules appear more universal than originally anticipated. The agreement of predicted hydration fractions for collagen and other proteins suggests water chains associated with proteins chains in the α-helix and β-pleat configurations. This was not previously known. Preliminary NMR titration studies of multiple proteins rich in both α-helix and β-pleat in our laboratories universally follow the “Ramachandran” hydration rules developed for collagen. We tentatively propose that globular proteins obey the collagen stoichiometric hydration rules of the collagen triple helix. The topic needs further investigation.
Funding for much of this work came from discretional funds supplied by Malcolm Jones Distinguished Professorship for which I am grateful to Drs. Reuter, Dodd and my other colleagues from the Radiology Department in San Antonio.
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Received 30 May 2005; accepted 30 September 2005doi:10.1016/j.cellbi.2005.09.009