Isotope Reverse-Labeled Infrared Spectroscopy as a Probe of In-Cell Protein Structure.

While recent years have seen great progress in determining the three-dimensional structure of isolated proteins, monitoring protein structure inside live cells remains extremely difficult. Here, we examine the utility of Fourier transform infrared (FTIR) spectroscopy as a probe of protein structure in live bacterial cells. Selective isotope enrichment is used both to distinguish recombinantly expressed NuG2b protein from the cellular background and to examine the conformation of specific residues in the protein. To maximize labeling flexibility and to improve spectral resolution between label and main-band peaks, we carry out isotope-labeling experiments in "reverse-labeling" mode: cells are initially grown in 13C-enriched media, with specific 12C-labeled amino acids added when protein expression is induced. 1 Because FTIR measurements require only around 20 µL of sample and each measurement takes only a few minutes to complete, isotope-labeling costs are minimal, allowing us to label multiple different residues in parallel in simultaneously grown cultures. For the stable NuG2b protein, isotope difference spectra from live bacterial cultures are nearly identical to spectra from isolated proteins, confirming that the structure of the protein is unperturbed by the cellular environment. By combining such measurements with site-directed mutagenesis, we further demonstrate that the local conformation of individual amino acids can be monitored, allowing us to determine, for example, whether a specific site in the protein contributes to α-helix or ß-sheet structures.

Controlling Vibronic Coupling in Chlorophyll Proteins: The Effects of Excitonic Delocalization and Vibrational Localization.

Vibrational-electronic (vibronic) coupling plays a critical role in excitation energy transfer in molecular aggregates and pigment-protein complexes (PPCs). But the interplay between excitonic delocalization and vibronic interactions is complex, often leaving even qualitative questions as to what conceptual framework (e.g., Redfield versus Förster theory) should be used to interpret experimental results. To shed light on this issue, we report here on the interplay between excitonic delocalization and vibronic coupling in site-directed mutants of the water-soluble chlorophyll protein (WSCP), as reflected in 77 K fluorescence spectra. Experimentally, we find that in PPCs where excitonic delocalization is disrupted (either by mutagenesis or heterodimer formation), the relative intensity of the vibrational sideband (VSB) in fluorescence spectra is suppressed by up to 37% compared to that of the native protein. Numerical simulations reveal that this effect results from the localization of high-frequency vibrations in the coupled system; while excitonic delocalization suppresses the purely electronic transition due to H-aggregate-like dipole-dipole interference, high-frequency vibrations are unaffected, leading to a relative enhancement of the VSB. By comparing VSB intensities of PPCs both in the presence and absence of excitonic delocalization, we extract a set of "local" Huang-Rhys (HR) factors for Chl a in WSCP. More generally, our results suggest a significant role for geometric effects in controlling energy-transfer rates (which depend sensitively on absorption/fluorescence line shapes) in molecular aggregates and PPCs.

Distinct electrostatic frequency tuning rates for amide I and amide I' vibrations.

Amide I spectroscopy probes the backbone C=O stretch vibrations of peptides and proteins. Amide I spectra are often collected in deuterated water (D2O) since this provides a cleaner background in the amide I frequency range; such data are often referred to as amide I' spectra since deuteration induces changes in the mode structure, including a roughly ∼10 cm-1 redshift. For biological samples, however, deuteration is often not possible. As amide I frequency maps are increasingly applied to quantitative protein structural analysis, this raises the interesting challenge of drawing direct connections between amide I and amide I' data. We here analyze amide I and amide I' peak frequencies for a series of dipeptides and related compounds. Changes in protonation state induce large electrostatic shifts in the peak frequencies, allowing us to amass a sizable library of data points for direct amide I/amide I' comparison. While we find an excellent linear correlation between amide I and amide I' peak frequencies, the deuteration-induced shift is smaller for more red-shifted vibrations, indicating different electrostatic tuning rates in the two solvents. H2O/D2O shifts were negligible for proline-containing dipeptides that lack exchangeable amide hydrogens, indicating that the intrinsic properties of the solvent do not strongly influence the H/D shift. These findings indicate that the distinct tuning rates observed for the two vibrations arise from modifications to the intrinsic properties of the amide bond and provide (at least for solvated dipeptides) a simple, linear "map" for translating between amide I and amide I' frequencies.

Accurate prediction of mutation-induced frequency shifts in chlorophyll proteins with a simple electrostatic model.

Photosynthetic pigment-protein complexes control local chlorophyll (Chl) transition frequencies through a variety of electrostatic and steric forces. Site-directed mutations can modify this local spectroscopic tuning, providing critical insight into native photosynthetic functions and offering the tantalizing prospect of creating rationally designed Chl proteins with customized optical properties. Unfortunately, at present, no proven methods exist for reliably predicting mutation-induced frequency shifts in advance, limiting the method's utility for quantitative applications. Here, we address this challenge by constructing a series of point mutants in the water-soluble chlorophyll protein of Lepidium virginicum and using them to test the reliability of a simple computational protocol for mutation-induced site energy shifts. The protocol uses molecular dynamics to prepare mutant protein structures and the charge density coupling model of Adolphs et al. [Photosynth. Res. 95, 197-209 (2008)] for site energy prediction; a graphical interface that implements the protocol automatically is published online at http://nanohub.org/tools/pigmenthunter. With the exception of a single outlier (presumably due to unexpected structural changes), we find that the calculated frequency shifts match the experiment remarkably well, with an average error of 1.6 nm over a 9 nm spread in wavelengths. We anticipate that the accuracy of the method can be improved in the future with more advanced sampling of mutant protein structures.