Linewidth protein

Table I reports the observed NMR linewidths for the H/3 protons of the coordinating cysteines in a series of iron-sulfur proteins with increasing nuclearity of the cluster, and in different oxidation states. We have attempted to rationalize the linewidths on the basis of the equations describing the Solomon and Curie contributions to the nuclear transverse relaxation rate [Eqs. (1) and (2)]. When dealing with polymetallic systems, the S value of the ground state has been used in the equations. When the ground state had S = 0, reference was made to the S of the first excited state and the results were scaled for the partial population of the state. In addition, in polymetallic systems it is also important to account for the fact that the orbitals of each iron atom contribute differently to the populated levels. For each level, the enhancement of nuclear relaxation induced by each iron is proportional to the square of the contribution of its orbitals (54). In practice, one has to calculate the following coefficient for each iron atom ...

When one of the Fe-coordinating Ns of the porphyrin is made inequivalent to the others, for example, by pulling on it, or by putting a protein structure around the cofactor, then the molecular x axis and y axis become inequivalent, and the axial EPR spectrum turns into the rhombic spectrum in trace d with derivative trace e (see also Table 5.4). There are now three features in the spectrum a peak, a zero crossing, and a negative peak, and their field positions closely (exactly for zero linewidth) correspond to those of the g-values, gx, gy, and gz. Finally, in trace f of Figure 5.4, which is the experimental X-band spectrum of cytochrome c, it can be seen that not only the g-value (peak position) but also the linewidth is frequently found to be anisotropic. This extra complication will be discussed extensively in Chapter 9. 

This chapter considers the distribution of spin Hamiltonian parameters and their relation to conformational distribution of biomolecular structure. Distribution of a g-value or g-strain leads to an inhomogeneous broadening of the resonance line. Just like the g-value, also the linewidth, W, in general, turns out to be anisotropic, and this has important consequences for powder patterns, that is, for the shape of EPR spectra from randomly oriented molecules. A statistical theory of g-strain is developed, and it is subsequently found that a special case of this theory (the case of full correlation between strain parameters) turns out to properly describe broadening in bioEPR. The possible cause and nature of strain in paramagnetic proteins is discussed. 

The magnetic susceptibility relaxation is usually more important for than for Ti. In fact, this mechanism is often dominant in determining the proton linewidth in paramagnetic proteins at high magnetic fields (3). Gillis and co-workers have recently developed a theory for the related case of proton linewidth in colloidal solutions of so-called superparamagnetic particles (54,55). 

A TOCSY experiment on a paramagnetic molecule is reported in Fig. 8.7 for the 5Cl-Ni-SAL-MeDPT complex [5]. Cross peaks between signals with linewidths of the order of 100 Hz were easily detected. In particular, the couplings of each aromatic proton with its neighbors are evident. TOCSY cross peaks between signals with similarly broad lines can also be detected in proteins (see Fig. 8.18) [24]. 

Such a decrease in the linewidth may result from a decrease in the Gd3+ coordination number upon formation of the macromolecular complex, which could result in greater symmetry and a lower zero-field splitting for the Gd3+ ion. This spectrum is independent of temperature between 4 and 25°C and is independent of the Gd3+/ ATPase ratio up to 2 Gd + ions/ATPase molecule. The peak-to-peak linewidth of 285 G sets a lower limit of 2,3 x 10"10s Qn the electron spin relaxation time of enzyme-bound Gd +t This symmetric, narrow EPR spectrum for the Gd3+-ATPase complex is compared in Figure 13B to that of Gd3+ bound to parvalbumin, a Ca2+-binding protein from carp. In this case, the spectrum is extremely broad and suggests a greatly distorted Gd3+ coordination geometry compared to the Ca2+-ATPase. 

The use of techniques that focus on a subset of resonances make it possible to do productive NMR experiments on systems that do not have the narrowest possible linewidths, and thus to investigate more challenging proteins or to optimize sample conditions for a particular functional state rather than for the narrowest resonances. However, since the information content of the NMR experiment depends on the number of resolvable resonances, which depends on their linewidths, it is critical to seek conditions that minimize the linewidths while preserving functionality. The membrane protein system of interest will dictate which sample types are possible and which conditions preserve functionality Table 1 documents membrane protein linewidths that have been observed in a variety of sample types including nanocrystals, 2D crystals, detergent micelles, proteoliposomes and nanodisks. 

TABLE 1 Linewidths of various membrane proteins under study by solid-state NMR... 

---