2D IR Probes Projects
NAD+ Analogs with Azide Labels
NAD+ (nicotinamide adenine dinucleotide) is a common redox cofactor that is used by many enzymes making it an attractive target to label with a chromophore for 2D IR spectroscopy because such a chromophore would have widespread applicability. We have prepared synthetic analogs of NAD+ with azide labels replacing the amide group on the nicotinamide ring. The first such label was azido-NAD+, in which the azide group is attached directly to the C3 position of the nicotinamide ring. This analog has the advantage of being as modest of a perturbation as we can make with this strategy, but it suffers from the fact that the molar absorptivity of the azide gorup in this position is quite low. As an alternative, we have also prepared PAAD+ (picolyl azide adenine dinucleotide) in which the azido group os separated from the nicotinamde ring by a methylene spacer. This system has the advantage that the molar absorptivity of the probe is much improved relative to azido-NAD+, but the larger functionality in place of the amide group is likely to cause a larger perturbation that will make this probe unsuitable for many applications. Nevertheless, the development of these probes suggests that this approach is a promising way to introduce infrared chromophores to study protein dynamics using 2D IR spectroscopy.
Several groups have reported an apporach to add a thiocyanate label to a protein by functionalizing cysteine. Because is is straightforward to selectively place single cysteine residues at almost any location in a protein by site-directed mutagenesis, this method for introducing a spectroscopic label is particularly attractive. The one drawback of this method is the fact that the alkyl thiocyanate has a particularly weak transition moment. Nevertheless, the attractive features of this probe: its sensitivity to electrostatic fields, relatively long population lifetime and, the fact that it can be incorporated in place of hydrophobic residuce such as isoleucine making very minor perturbations to the protein, make cyanylated cysteines a natural target for applications involving 2D IR spectroscopy. In spite of the challenges associated with using this weak probe, we have recently made improvements to our apparatus that make measurements using this probe accessible. The signals are so weak that they are of a similar magnitude to the background response of water so that we have to measure and subtract this background response to isolate the SCN signal. Nevertheless, we are excited by the opportunities that this probe will make possible to address interesting protein systems that do not have an intrinsic IR chromophore that is suitable for studying protein dynamics.