muramidase and monobromobimane

We have determined the structural changes that accompany the formation of a stable complex between a destabilized mutant of T4 lysozyme (T4L) and the small heat shock protein alpha-crystallin. Using pairs of fluorescence or spin label probes to fingerprint the T4L tertiary fold, we demonstrate that binding disrupts tertiary packing in the two domains as well as across the active-site cleft. Furthermore, increased distances between i and i+4 residues of helices support a model in which the bound structure is not native-like but significantly unfolded. In the confines of the oligomer, T4L has a preferential orientation with residues in the more hydrophobic C-terminal domain sequestered in a buried environment, while residues in the N-terminal domain are exposed to the aqueous solvent. Furthermore, electron paramagnetic resonance spectral line shapes of sites in the N-terminal domain are narrower than in the folded, unbound T4L reflecting an unstructured backbone and an asymmetric pattern of contacts between T4L and alpha-crystallin. The net orientation is not affected by the location of the destabilizing mutation consistent with the notion that binding is not triggered by recognition of localized unfolding. Together, the structural and thermodynamic data indicate that the stably bound conformation of T4L is unfolded and support a model in which the two modes of substrate binding originate from two discrete binding sites on the chaperone.

Topics: alpha-Crystallins; Bacteriophage T4; Binding Sites; Bridged Bicyclo Compounds; Cloning, Molecular; Cyclic N-Oxides; Escherichia coli; Fluorescent Dyes; Heat-Shock Proteins, Small; Hydrophobic and Hydrophilic Interactions; Models, Biological; Muramidase; Mutation; Protein Binding; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Solvents; Spectrometry, Fluorescence; Spin Labels; Substrate Specificity; Thermodynamics; Water

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix-turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of <40 A(2)), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, DeltaDeltaG values of < or = 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation lambda(max), emission lambda(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission lambda(max) and the steady-state anisotropy values most accurately reflect the solvent surface accessibility at each site as calculated from the crystal structure of cysteine-less T4 lysozyme. The parameters we identify allow the classification of each site as buried, partially buried, or exposed. We find that the variations in these parameters as a function of residue number reflect the sequence-specific secondary structure, the determination of which is a key step for modeling a protein of unknown structure.

Topics: Bacteriophage T4; Bridged Bicyclo Compounds; Enzyme Stability; Fluorescence Polarization; Fluorescent Dyes; Muramidase; Mutagenesis, Site-Directed; Protein Conformation; Protein Folding; Protein Structure, Secondary; Quantum Theory; Solvents; Spectrometry, Fluorescence; Thermodynamics