NMR relaxation experiments can provide information on overall and internal motions in proteins. This review consists of a concise report on the evolution of the theories for nuclear relaxation followed by an overview of mathematical models for internal motions in proteins. Next, the method of spectral density mapping with recent developments is reviewed. This is followed by a discussion of pulse sequences for relaxation experiments. Finally, we review recent studies correlating relaxation parameters, in particular spectral density functions, with structural features of proteins and with results of molecular dynamics simulations.

Here we investigate the effects of the naturally occurring threonine-linked L-fucose moiety on the structure, dynamics and stability of the proteinase inhibitor PMP-C (Pars intercerebralis major peptide C). The three-dimensional structure of PMP-C fucosylated on Thr 9 has been determined by NMR spectroscopy and simulated annealing. The fucose ring is very well ordered, held in place by hydrophobic and hydrogen bond interactions with Thr 16 and Arg 18. Comparing the NMR data and the structure of the fucosylated inhibitor with those of the nonfucosylated form shows that conformational changes only occur in the vicinity of the fucose moiety. Nevertheless, a comparative analysis of the exchange rates of amide protons indicates that fucosylation is responsible for an overall decrease of the dynamic fluctuations of the molecule. This correlates well with an increase in stability of approximately 1 kcal mol-1 as monitored by thermal denaturation.

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Recombinant production of HPV oncoprotein E6 is notoriously difficult. The unfused sequence is produced in inclusion bodies. By contrast, fusions of E6 to the C-terminus of carrier proteins such as maltose-binding protein or glutathione-S-transferase are produced soluble. However, it has not yet been possible to purify E6 protein from such fusion constructs. Here, we show that this was due to the biophysical heterogeneity of the fusion preparations. We find that soluble MBP-E6 preparations contain two subpopulations. A major fraction is aggregated and contains exclusively misfolded E6 moieties ('soluble inclusion bodies'). A minor fraction is monodisperse and contains the properly folded E6 moieties. Using monodispersity as a screening criterion, we optimized the expression conditions, the purification process and the sequence of E6, finally obtaining stable monodisperse MBP-E6 preparations. In contrast to aggregated MBP-E6, these preparations yielded fully soluble E6 after proteolytic removal of MBP. Once purified, these E6 proteins are stable, folded and biologically active. The first biophysical measurements on pure E6 were performed. This work shows that solubility is not a sufficient criterion to check that the passenger protein in a fusion construct is properly folded and active. By contrast, monodispersity appears as a better quality criterion. The monodispersity-based strategy presented here constitutes a general method to prepare fusion proteins with optimized folding and biological activity.