the second harmonic (527 nm) of a Nd:YLF laser operating at 100 MHz. The dye laser contained a single plate birefringent filter tuned to produce laser oscillation at 610 nm and a pulse width of approximately 1 ps full width at half maximum (FWHM). The dye laser was cavity-dumped at 1 MHz, and then frequency doubled using a potassium dihydrogen phosphate nonlinear crystal. Sample fluorescence was spectrally filtered with a monochromator (bandpass ∼ 10 nm) and detected with a cooled microchannel plate (PMT, Hamamatsu R2809U-11). Fluorescence transients were measured at the magic angle, 54.7 ° [24]. Temporal response function of the system was measured to a FWHM of ca. 50 ps.
Construction of QD-Protein Conjugate Model: A model for the three-dimensional conformation of Cy3-maleimide was constructed using Chem-3D Ultra 8.0 (CambridgeSoft, Cambridge, MA). Using energy minimization MM2, low-energy conformers were located using the MM2 molecular dynamics module within Chem-3D Ultra. Conformer energies ranged from 17.9 to 23.0 kcal mol -1 . A low-energy conformer was selected, which had the maleimide oriented such that it could interact with a cysteine side chain on the protein to form a covalent bond. The Cy3-maleimide model was merged with the X-ray crystal structure of MBP using MidasPlus [16,25]. The preferred orientation of QD-surface-located MBP was previously investigated [16]. An optimal configuration of the QD-MBP-His assembly is assumed in the present analysis. Fitting of the open and closed forms of MBP was performed within MidasPlus by aligning six a carbons of residues in one of the two MBP lobes in the structure. Distance between the Cy3 fluorophore and the QD center was 73.7 Å corresponding to the previously ascertained value [16].