Bimolecular quenching of tryptophan fluorescence in a membrane protein: Evolution of local solvation and environment during folding into a bilayer.

Fluorescence spectroscopy, including Stern-Volmer quenching, is a valuable tool for the study of protein dynamics. Changes in protein solvation during the folding reaction of a membrane protein, Outer membrane protein A (OmpA), into lipid bilayers was probed with bimolecular fluorescence quenching with acrylamide quencher. Six single-tryptophan OmpA mutants (W7, W15, W57, W102, W129, and W143) allowed for site-specific investigations at varying locations within the transmembrane ß-barrel domain. A sphere-of-action quenching model that combines both static and dynamic components gave rise to Stern-Volmer quenching constants, KD, for OmpA denatured in 8.0 M urea, aggregated in 0.5 M urea, adsorbed onto small unilamellar vesicles (SUVs), and folded in SUVs (t = 6 hrs). The average KD values were KDdenatured(6.4M-1)>KDaggregated5.9M-1>KDadsorbed(1.9M-1)>KDfolded(0.6M-1). With knowledge of the fluorescence lifetimes in the absence of quencher, the bimolecular quenching constants, kq, were derived; the evolution of kq (and therefore KD)during the folding reaction into SUVs (t = 0 hr to t = 6 hrs) revealed desolvation timescales, τdesolv of 41-46 min (W7, W15, W57, W102), 27 min (W129), and 15 min (W143). The evolution of λmax during folding revealed fast and slow components, τenvironmentfast and τenvironmentslow of 7-13 min and 25-84 min, respectively, for all mutants. For the five lipid- facing mutants (W7, W15, W57, W129, and W143), the general trend was τenvironmentfast7-13min<τdesolv15-46min≤τenvironmentslow(25-84min). These results suggest that there is an initial fast step in which there is a large change in polarity to a hydrophobic environment, followed by a slower desolvation process during evolution within the hydrophobic environment. These results complement previous mechanisms of concerted folding and provide insights into site-specific changes in solvation during formation of native ß-barrel structure.

The structural basis for monoclonal antibody 5D2 binding to the tryptophan-rich loop of lipoprotein lipase.

For three decades, the LPL-specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

Folding of the ß-Barrel Membrane Protein OmpA into Nanodiscs.

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated ß-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm-1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.

UV Resonance Raman Spectroscopy as a Tool to Probe Membrane Protein Structure and Dynamics.

Ultraviolet resonance Raman (UVRR) spectroscopy is a vibrational technique that reveals structures and dynamics of biological macromolecules without the use of extrinsic labels. By tuning the Raman excitation wavelength to the deep UV region (e.g., 228 nm), Raman signal from tryptophan and tyrosine residues are selectively enhanced, allowing for the study of these functionally relevant amino acids in lipid and aqueous environments. In this chapter, we present methods on the UVRR data acquisition and analysis of the tryptophan vibrational modes of a model ß-barrel membrane protein, OmpA, in folded and unfolded conformations.