Elucidation of intermediate (mobile) and slow (solid-like) protein motions in bovine lens homogenates by carbon-13 NMR spectroscopy

Abstract

The motional dynamics of lens cytoplasmic proteins present in calf lens homogenates were investigated by two 13C nuclear magnetic resonance (NMR) techniques sensitive to molecular motion to further define the organizational differences between the cortex and nucleus. For the study of intermediate (mobile) protein rotational reorientation motion time scales [rotational correlation time (tau 0) range of 1-500 ns], we employed 13C off-resonance rotating frame spin-lattice relaxation, whereas for the study of slow (solidlike) motions (tau 0 greater than or equal to 10 microseconds) we used the solid-state NMR techniques of dipolar decoupling and cross-polarization. The frequency dependence of the peptide bond carbonyl off-resonance rotating frame spectral intensity ratio of the lens proteins present in native calf nuclear homogenate (42% protein) at 35 degrees C indicates the presence of a polydisperse mobile protein fraction with a tau 0,eff (mean) value of 57 ns. This mean value is consistent with the average value calculated from the known water-soluble nuclear lens protein polydispersity assuming a cytoplasmic viscosity 3 times that of pure water. Lowering the temperature to 1 degree C, a temperature which produces the cold cataract, results in an overall decrease in tau 0,eff to 43 ns, suggesting a selective removal of beta H-, LM-, and possibly gamma s-crystallins from the mobile lens protein population. The presence of solidlike or motionally restricted protein species was established by dipolar decoupling and cross-polarization. The fraction of motionally restricted protein in the nuclear region varied from 0.35 to 0.45 in the temperature range of 35-1 degree C. For native cortical homogenate (25% protein), the off-resonances rotating frame spectral intensity ratio frequency-dependent curves for the protein carbonyl resonance yielded tau 0,eff values of 34 and 80 ns at 35 and 1 degree C, respectively. Both values were reconciled with the known lens cortex soluble protein polydispersity using an assumed cytoplasmic viscosity 1.5 times that of pure water at the same temperature. Comparison of proton dipolar-decoupled and nondecoupled 13C NMR spectra of native cortical homogenate at 20 degrees C indicates the absence of significant contributions from slowly tumbling, motionally restricted species. This interpretation was confirmed by the failure to detect significant lens protein 13C-1H cross-polarization at this temperature. However, at 1 degree C, the fraction of solidlike protein was 0.15. Concentrated cortical homogenates at 20 degrees C (42% protein), by contrast, gave cross-polarization spectra with maximum absolute signal intensities 50-70% of native nuclear homogenates, but with similar magnetization parameters...

An Impediment to Glutathione Diffusion in Older Normal Human Lenses: a Possible Precondition for Nuclear Cataract

“…NMR studies have shown that the protein phases present in cortex and nucleus of the lens may differ (Morgan et al, 1989). We have preliminary evidence using nuclear magnetic resonance imaging (NMRI) to elucidate long and short range D # O diffusion within the lens, which suggests that the barrier which develops to GSH may also function in the case of other low molecular weight compounds (Moffat et al, 1998).…”

Characterization of Covalent Multimers of Crystallins in Aging Human Lenses

“…The complex showed the presence of two tryptic peptide sequences of ␣A-crystallin: TLGPFYPSR (residues 13-21) and HFSPEDLTVK (residues 79 -88), and six tryptic peptides of ␣B-crystallin with sequences of: APSWFDTGLSEMR (oxidized Met residues 57-69), APSWFDTGLSEMRLEK (residue 57-72), HFSPEELK (residues 83-90), DRFSVNLDVK (residues 73-82), QDEHGFISR (residues 108 -116), and IPAD-VDPLTITSSLSSDGVLTVNGPR (residues 124 -149). This spot also contained three ␤A3-crystallin tryptic peptides with the sequences of ITIYDQENFQGKR (residues 33-45), WDAWSG-SNAYHIER (residues 96 -109), and MTIFEKENFIGR (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), two ␤A4-crystallin tryptic peptides with the sequences of MVVWDEDGFQGR (oxidized Met, residues 13-24) and LTIFEQENFLGK (residues 107-118), and four ␤B1 tryptic peptides with the sequence of LVVFELENFQGR (residues 60 -71), VSSGTWVGYQYPGYR (residues 187-201), ISLFE-GANFK (residues 151-160), and GYQYLLEPGDFR (residues 202-213). Additional peptides in the complex were one ␤B2 tryptic peptide with the sequence of VQSGTWVGYQYPGYR (residues 145-159), four ␥S tryptic fragments with the sequence of ITFYEDKNFQGR (residues 7-18), AVHLPSGGQYK (residues 84 -94), QYLLDKK (residues 148 -154), and KPIDW-GAASPAVQSFR (residues 158 -173), and one tryptic peptide of ␥D-crystallin with the sequence of QYLLMPGDYR (with oxidized Met, residues 143-152).…”

“…Additionally, the cross-linked species might be represented by ␣-crystallin lens membrane (18 -21), and ␣-crystallin intermediate filament proteins complexes (22). Together, these complexes may represent the supramolecular species in the lens, previously identified in vivo as 0.12-to 0.9-m particles with an estimated molecular mass of 2 ϫ 10 9 Da by NMR spectroscopy (23) and by quasielastic light scattering (24).…”

Determination of proton magnetization transfer rate constants in heterogeneous biological systems

“…The transfer of proton magnetization between the macromolecular and solvent (water) components of tissue has generally been modelled phenomenologically in terms of two spin baths representing solid-like or immobile macromolecular protons and mobile solvent protons (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). To accommodate the complexities of the ocular lens, a tissue that includes both moderately mobile (7, -100 ns) and solid-like protein macromolecular components (18,19), Kuwata et al (16) extended the two spin bath formalism to include two macromolecular proton spin baths plus a solvent spin bath. Moreover, this model of magnetization transfer was cast in the form of a matrix formalism enabling the generalization to N-proton spin baths.…”

“…To make a direct comparison of the magnetization transfer rate constant obtained by assuming total saturation of the solid-like spin bath and by a formalism that does not require this assumption (16), we have chosen two systems: a model system of cross-linked bovine serum albumin, which has been assumed to consist of two proton spin baths (macromolecular and water) (3,14) and (101, K (141, RAE (161, and RAC (16). 331 the nuclear region of the ocular lens, which represents a tissue system containing three spin baths (two macromolecular + water) (16,18,19).…”