The dynamics of the CN --ligated ferric cytochrome c (CytcCN) in D2O at 283 K following Q-band photoexcitation at 575 nm was observed using femtosecond time-resolved vibrational spectroscopy. The equilibrium vibrational spectrum of the CN stretching mode of CytcCN shows two overlapping bands: one main band (82%) at 2122 cmwith 23 cm -1 full width at half maximum (fwhm) and the other band (18%) at 2116 cm -1 with 7 cm -1 fwhm. The timeresolved spectra show bleaching of the CN fundamental mode of CytcCN and two absorption features at lower energies. The bleach signal and both absorption features are all formed within the time resolution of the experiment (< 200 fs) and decay with a life time of 1.9 ps. One transient absorption feature, appearing immediately red to the bleach signal, results from the thermal excitation of low-frequency modes of the heme that anharmonically couple to the CN fundamental mode, thereby shifting the CN mode to lower energies. The shift of the CN mode decays with a lifetime of 2 ps, equivalent to the time scale for vibrational cooling of the low-frequency heme modes. The other transient absorption feature, which is 3.3 times weaker than the bleach signal and shifted 27 cm -1 toward lower energies, is attributed to the CN mode in an electronically excited state where the CN bond is weakened with a lowered extinction coefficient. These observations suggest that photoexcited CytcCN mainly undergoes ultrafast radiationless relaxation, causing photo-deligation of CN -from CytcCN highly inefficient. As also observed in CN --ligated myoglobin, inefficient ligand photodissociation might be a general property of CN --ligated ferric hemes.
Fluorescence correlation spectroscopy (FCS) is a sophisticated and an accurate analytical technique used to study the diffusion of molecules in a solution at the single-molecule level. FCS is strongly affected by many factors such as the stability of the excitation power, photochemical processes, mismatch between the refractive indices, and variations in the cover glass thickness. We have studied FCS near the surface of a cover glass by using rhodamine 123 as a fluorescent probe and have observed that the surface has a strong influence on the measurements. The temporal autocorrelation of FCS decays with two characteristic times when the confocal detection volume is positioned near the surface of the cover glass. As the position of the detection volume is moved away from the surface, the FCS autocorrelation becomes one-component decaying; the characteristic time of the decay is the same as the faster-decaying component in the FCS autocorrelation near the surface. This observation suggests that the faster component can be attributed to the free diffusion of the probe molecules in the solution, while the slow component has its origin from the interaction between the probe molecules and the surface. We have characterized the surface contribution to the FCS measurements near the surface by changing the position of the detection volume relative to the surface. The influence of the surface on the diffusion of the probe molecules was monitored by changing the chemical properties of the surface. The surface contribution to the temporal autocorrelation of the FCS strongly depends on the chemical nature of the surface. The hydrophobicity of the surface is a major factor determining the surface influence on the free diffusion of the probe molecules near the surface.
ABSTRACT. Fluorescence correlation spectroscopy (FCS) is an emerging fluorescence technique used to study the dynamics of proteins on a millisecond to microsecond time scale at the single-molecule level. Solution pH-modulated protein conformational changes can be manifested by binding rate, fluorescence lifetime, and binding specificity of a probe molecule. The fluorescence lifetime of Nile red (NR) bound to apomyoglobin (apoMb) was measured to be 6 ± 0.3 ns, much longer than that in water solution (2.9 ± 0.2 ns). As the unfolding population of apoMb increased by lowering pH of solution, the fraction for the longer lifetime of NR decreased with an increasing fraction for the shorter lifetime of NR in water. Unlike 1-anilino-8-naphthalene sulfonic acid, which has many lifetimes due to nonspecific binding to the unfolded apoMb, NR bound to apoMb possesses only a single lifetime. These results suggest that NR binds specifically to native apoMb and thus can be utilized to probe the folding/unfolding dynamics of apoMb using FCS.
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