In This Issue

1587 Probing local structural fluctuations in myoglobin by size-dependent thiol-disulfide exchange

Margaret M. Stratton, Thomas A. Cutler, Jeung-Hoi Ha, and Stewart N. Loh

Hydrogen exchange (HX) is an invaluable tool for identifying partially unfolded protein conformations that are invisible to nearly all other techniques. Buried amide groups can exchange their hydrogens with those of water only once that region becomes exposed to the solvent. One of the limitations of HX, however, is that molecules that probe for opening events are limited to hydrogen isotopes of water. Thiol-disulfide exchange (SX) complements HX in that probes of different chemical composition can be employed. Stratton et al. use thiosulfonate groups of different sizes to characterize, for the first time, the sizes of local structural unfolding, or “breathing” events, that expose buried cysteine side chains to exchange in myoglobin.

1544 Protein denaturation and protein:drug interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Matthieu Gaudet, Nina Remtulla, Sophie E. Jackson, Ewan R. G. Main, Daniel G. Bracewell, Gabriel Aeppli, and Paul A. Dalby

In this report, Gaudet et al. present a nanolitre scale technique in micro-capillaries to measure intrinsic protein fluorescence and obtain accurate protein denaturation curves at equilibrium. Free energies of unfolding were determined for the first time by such a method at this scale, and used to determine the affinity of the immunosuppressant rapamycin to the cellular immunophilin FKBP12, and also from a double mutant cycle analysis, the interaction energy between rapamycin and the Phe99 residue of FKBP-12. The technique has significant potential, by coupling to microfluidic devices for sample preparation, to decrease the sample requirements for drug discovery, diagnostics and protein stability characterization.

1555 Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Magnus Kjaergaard, Ann-Beth Nørholm, Ruth Hendus-Altenburger, Stine F. Pedersen, Flemming M. Poulsen, and Birthe B. Kragelund

Intrinsically disordered proteins undergo temperature dependent conformational transitions. Due to a lack of high resolution data the exact nature of these have remained unknown. Using multidimensional NMR spectroscopy, Kjaergaard et al. show that the populations of transiently formed α-helices decrease with increasing temperature. This conclusion is opposite to what had previously been inferred from circular dichroism spectroscopy. The discrepancy is caused by a temperature-dependent redistribution of the random coil, which results in a more compact ensemble at higher temperatures.

1490 Circularly permuted monomeric red fluorescent proteins with new termini in the β-sheet

Haley J. Carlson, Darrel W. Cotton, and Robert E. Campbell

Circularly permuted versions of green fluorescent proteins (FPs) are an important class of tools in live cell fluorescence biosensing applications. However, there have been very few reports of using circularly permuted red FPs as components of biosensors, despite the potential advantage associated with the longer emission wavelength. A major factor limiting the use of circularly permuted red FPs has been the relatively poor brightness of reported variants. To address this limitation, we have subjected a circularly permuted red FP to directed evolution to produce a variant with brightness comparable to that of popular non-permuted red FPs.