Determination of protein secondary structure and solvent accessibility using site-directed fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane

Steven E. Mansoor, Hassane S. Mchaourab, David L. Farrens

Research output: Contribution to journalArticlepeer-review

52 Scopus citations

Abstract

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 Å2), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, ΔΔG values of ≤ 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation λ(max), emission λ(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission λ(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.

Original languageEnglish (US)
Pages (from-to)16383-16393
Number of pages11
JournalBiochemistry
Volume38
Issue number49
DOIs
StatePublished - Dec 7 1999

ASJC Scopus subject areas

  • Biochemistry

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