Role of the Triplet State and Protein Dynamics in the Formation and Stability of the Tryptophan Radical in an Apoazurin Mutant.

The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron acceptor, the neutral radical (W48•) is formed. The lifetime of W48• in apoazurin is 41 s, which is shorter than the lifetime of several hours in Zn-substituted azurin. Molecular dynamics simulations revealed enhanced fluctuations of apoazurin which likely destabilize W48•. The photophysics of W48 was investigated to probe the precursor state for ET. The phosphorescence intensity was eliminated in the presence of an electron acceptor while the fluorescence was unchanged; this quenching of the phosphorescence is attributed to ET. The kinetics associated with W48• were examined with a model that incorporates intersystem crossing, ET, deprotonation, and decay of the cation radical. The estimated rate constants for ET (6 × 106 s-1) and deprotonation (3 × 105 s-1) are in agreement with a photoinduced mechanism where W48• is derived from the triplet state. The triplet as the precursor state for ET was supported by photolysis of apoazurin with 280 nm in the absence and presence of triplet-absorbing 405 nm light. Absorption bands from the neutral radical were observed only in the presence of blue light.

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.

Insights into Protein Structure and Dynamics by Ultraviolet and Visible Resonance Raman Spectroscopy.

Raman spectroscopy is a form of vibrational spectroscopy based on inelastic scattering of light. In resonance Raman spectroscopy, the wavelength of the incident light falls within an absorption band of a chromophore, and this overlap of excitation and absorption energy greatly enhances the Raman scattering efficiency of the absorbing species. The ability to probe vibrational spectra of select chromophores within a complex mixture of molecules makes resonance Raman spectroscopy an excellent tool for studies of biomolecules. In this Current Topic, we discuss the type of molecular insights obtained from steady-state and time-resolved resonance Raman studies of a prototypical photoactive protein, rhodopsin. We also review recent efforts in ultraviolet resonance Raman investigations of soluble and membrane-associated biomolecules, including integral membrane proteins and antimicrobial peptides. These examples illustrate that resonance Raman is a sensitive, selective, and practical method for studying the structures of biological molecules, and the molecular bonding, geometry, and environments of protein cofactors, the backbone, and side chains.

Förster resonance energy transfer as a probe of membrane protein folding.

The folding reaction of a ß-barrel membrane protein, outer membrane protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.

Kinetic spectroscopy of heme hydration and ligand binding in myoglobin and isolated hemoglobin chains: an optical window into heme pocket water dynamics.

The entry of a water molecule into the distal heme pocket of pentacoordinate heme proteins such as myoglobin and the alpha,beta chains of hemoglobin can be detected by time-resolved spectroscopy in the heme visible bands after photolysis of the CO complex. Reviewing the evidence from spectrokinetic studies of Mb variants, we find that this optical method measures the occupancy of non(heme)coordinated water in the distal pocket, n(w), with high fidelity. This evidence further suggests that perturbation of the kinetic barrier presented by distal pocket water is often the dominant mechanism by which active site mutations affect the bimolecular rate constant for CO binding. Water entry into the heme pockets of isolated hemoglobin subunits was detected by optical methods. Internal hydration is higher in the native alpha chains than in the beta chains, in agreement with previous crystallographic results for the subunits within Hb tetramers. The kinetic parameters obtained from modeling of the water entry and ligand rebinding in Mb mutants and native Hb chains are consistent with an inverse dependence of the bimolecular association rate constant on the water occupancy factor. This correlation suggests that water and ligand mutually exclude one another from the distal pockets of both types of hemoglobin chains and myoglobin.