Richard J. Wittebort

Quantitative Observation of Backbone Disorder in Native Elastin

Elastin is a key protein in soft tissue function and pathology. Establishing a structural basis for understanding its reversible elasticity has proven to be difficult. Complementary to structure is the important aspect of flexibility and disorder in elastin. We have used solid-state NMR methods to examine polypeptide and hydrate ordering in both elastic (hydrated) and brittle (dry) elastin fibers and conclude (i) that tightly bound waters are absent in both dry and hydrated elastin and (ii) that the backbone in the hydrated protein is highly disordered with large amplitude motions. The hydrate was studied by 2H and 17O NMR, and the polypeptide by 13C and 2H NMR. Using a two-dimensional 13C MAS method, an upper limit of S < 0.1 was determined for the backbone carbonyl group order parameter in hydrated elastin. For comparison, S ∼∼ 0.9 in most proteins. The former result is substantiated by two additional observations: the absence of the characteristic 2H spectrum for stationary amides and “solution-like” 13C magic angle spinning spectra at 75 °C, at which the material retains elasticity. Comparison of the observed shifts with accepted values for α-helices, β-sheets, or random coils indicates a random coil structure at all carbons. These conclusions are discussed in the context of known thermodynamic properties of elastin and, more generally, protein folding. Because coacervation is an entropy-driven process, it is enhanced by the observed backbone disorder, which, we suggest, is the result of high proline content. This view is supported by recent studies of recombinant elastin polypeptides with systematic proline substitutions.

Backbone motions in a crystalline protein from field-dependent 2H-NMR relaxation and line-shape analysis.

We have used 2H-nmr to study backbone dynamics of the 2H-labeled, slowly exchanging amide sites of fully hydrated, crystalline hen egg white lysozyme. Order parameters are determined from the residual quadrupole coupling and values increase from S2 = 0.85 at 290 K to S2 = 0.94 at 200 K. Dynamical rates are determined from spin-lattice relaxation at three nmr frequencies (38.8, 61.5, and 76.7 MHz). The approach used here is thus distinct from solution nmr studies where dynamical amplitudes and rates are both determined from relaxation measurements. At temperatures below 250 K, relaxation is independent of the nmr frequency indicating that backbone motions are fast compared to the nmr frequencies. However, as the temperature is increased above 250 K, relaxation is significantly more efficient at the lowest frequency, which shows, in addition, the presence of motions that are slow compared to the nmr frequencies. Using the values of S2 determined from the residual quadrupole coupling and a model-free relaxation formalism that allows for fast and slow internal motions, we conclude that these slow motions have correlation times in the range of 0.1 to 1.0 microsecond and are effectively frozen out at 250 K where fast motions of the amide planes with approximately 15 ps effective correlation times and 9 degrees rms amplitudes dominate relaxation. The fast internal motions increase slightly in amplitude as the temperature rises toward 290 K, but the correlation time, as is also observed in solution nmr studies of RNase H, is approximately constant. These findings are consistent with hypotheses of dynamic glass transitions in hydrated proteins arising from temperature-dependent damping of harmonic modes of motion above the transition point.

Effect of a Libration or Hopping Motion of theη2-Dihydrogen Ligand on Longitudinal NuclearMagnetic Resonance Relaxation

In the past, H—H distances of the η2‐dihydrogen ligand in transition metal complexes have been determined in the solid state by crystallographic or NMR studies (dH2cryst) and have been estimated in solution from 1JHD coupling constants (dH2HD) or minimum T1 values (dH2slow for slow internal motion of the H2 or dH2fast for fast spinning of a free rotor H2 where dH2fast=0.793dH2slow) as determined by 1H NMR. The best estimate of the H—H distance in solution was found to be dH2HD. This work shows that dH2HD is found to lie between dH2slow and dH2fast for many dihydrogen complexes reported in the literature. In certain cases this will be true if the correlation time of the H2 is similar to that of the molecular complex. Two other cases are considered here for the first time: (1) torsional oscillation of the H2 in a twofold potential energy surface and (2) hydrogens undergoing rapid 90° hops between sites of unequal population in a potential surface with a fourfold component. The 1JHD and T1min data from the literature for 73 dihydrogen complexes are examined in light of these two other possible cases. Dihydrogen in fast rotation is proposed for 32 complexes. Six complexes appear to have an H2 ligand with slow internal motion. Either torsional libration or fast hopping might have a significant influence on the T1 relaxation of the remaining 35 complexes.

2Q NMR of 2H2O ordering at solid interfaces

Solvent ordering at an interface can be studied by multiple-quantum NMR. Quantitative studies of (2)H2O ordering require clean double-quantum (2Q) filtration and an analysis of 2Q buildup curves that accounts for relaxation and, if randomly oriented samples are used, the distribution of residual couplings. A pulse sequence with absorption mode detection is extended for separating coherences by order and measuring relaxation times such as the 2Q filtered T2. Coherence separation is used to verify 2Q filtration and the 2Q filtered T2 is required to extract the coupling from the 2Q buildup curve when it is unresolved. With our analysis, the coupling extracted from the buildup curve in (2)H2O hydrated collagen was equivalent to the resolved coupling measured in the usual 1D experiment and the 2Q to 1Q signal ratio was in accord with theory. Application to buildup curves from (2)H2O hydrated elastin, which has an unresolved coupling, revealed a large increase in the 2Q signal upon mechanical stretch that is due to an increase in the ordered water fraction while changes in the residual coupling and T2 are small.

Order, Disorder, and Temperature-Driven Compaction in a Designed Elastin Protein

Artificial minielastin constructs have been designed that replicate the structure and function of natural elastins in a simpler context, allowing the NMR observation of structure and dynamics of elastin-like proteins with complete residue-specific resolution. We find that the alanine-rich cross-linking domains of elastin have a partially helical structure, but only when capped by proline-rich hydrophobic domains. We also find that the hydrophobic domains, composed of prominent 6-residue repeats VPGVGG and APGVGV found in natural elastins, appear random coil by both NMR chemical shift analysis and circular dichroism. However, these elastin hydrophobic domains exhibit structural bias for a dynamically disordered conformation that is neither helical nor β sheet with a degree of nonrandom structural bias which is dependent on residue type and position in the sequence. Another nonrandom-coil aspect of hydrophobic domain structure lies in the fact that, in contrast to other intrinsically disordered proteins, these hydrophobic domains retain a relatively condensed conformation whether attached to cross-linking domains or not. Importantly, these domains and the proteins containing them constrict with increasing temperature by up to 30% in volume without becoming more ordered. This property is often observed in nonbiological polymers and suggests that temperature-driven constriction is a new type of protein structural change that is linked to elastins biological functions of coacervation-driven assembly and elastic recoil.