Jimenez Lab

Femtosecond spectroscopy of biological cofactors

We are employing techniques developed for non-biological systems, and developing new techniques, for characterizing the dynamics of proteins with imbedded cofactors. An advantage of employing endogenous cofactors as a probe (i.e. rather than surface labeling with fluorophores) is that we can obtain information on native dynamics in the interior of the protein. However, one trade-off is that instead of having a choice of chromophores with desirable photophysical properties, one must incorporate the effects of complex intramolecular dynamics within the femtosecond signals.

One technique that has been very useful for studying chromophore-containing proteins is three-pulse photon echo peak shift spectroscopy (3PEPS). This four-wave mixing experiment is a resonant electronic spectroscopy employing three femtosecond pulses. Here is a diagram of the experiment:

The peak shift is half of the delay between the maximum intensities of the two echo signals (measured for a -scan), plotted as a function of T. Under common (but not all!) conditions, this peak shift resolves the time-correlation function, M(t), of the electronic transition frequency ( eg):

which may be illustrated in terms of the ground-excited state energy gap as follows:

For two-level chromophores, measurements of the peak shift decay provide information on the timescales of chromophore vibrations and protein motion. Furthermore, 3PEPS also resolves the inhomogeneous broadening of the system (a distribution of i), which is very useful because this broadening arises from disorder (e.g. multiple conformations). The frequency range of motions resolved by 3PEPS spans a very wide range: often from 500 cm-1 to 0.001 cm-1.

Many biological chromophores, such as hemes, are not well-approximated as two-level systems, and a variety of new phenomena must be considered when analyzing their echo signals. In particular, hemes, as do all porphyrins, have degenerate or nearly-degenerate excited state levels, as a consequence of their nearly-square planar symmetry. A ~ 100-300 cm-1 splitting between levels is caused by the symmetry-lowering effects of substituents on the porphyrin macrocycle, conformational distortions, or electric fields within a protein environment. A femtosecond pulse will simultaneously excite both levels. Secondly, since the excited state levels are orthogonal, polarization effects must be considered. Third, the symmetry properties of the vibrations must be considered: some vibrations cause both excited state levels to fluctuate in a correlated manner, and some vibrations cause the splitting itself to fluctuate. Finally, internal conversion from the initially excited states (and within the Soret band) to lower-lying states must be considered.

We used pulses from the second harmonic of a cavity-dumped Ti:sapphire oscillator to perform 3PEPS measurements on folded and denatured Zn-substituted cytochrome c, which binds Zn-protoporphyrin XI (shown above) as the co-factor. Interestingly, the dynamics of the unfolded protein are quite different: it is much "floppier" and more disordered than the native conformer. Here is a comparison of the data (only the first 1000 femtoseconds is shown. For more information, see our April 2006 publication in J. Chem. Phys):

Numerical simulations of the peak shift decay that incorporate all of the phenomena described, above at some level, show the peak shift timescales largely reflect the dynamics observed in two level systems. At present, we conclude that measurements on hemes are able to provide as much information on protein dynamics as measurements on simpler chromophores, if the internal conversion timescales and excited-state level splitting are known from separate experiments. We are currently exploring whether more sophisticated experiments are able to reveal more detailed information such as whether a particular time scale of fluctuation leads to correlated or uncorrelated motion of the excited state levels. We are also exploring the sensitivity of 3PEPS to the locations of site-directed mutations made within a protein.