A Co-Translationally Insertable Donor-Acceptor Pair for the Real Time Study of Vibrational Energy Transfer in Proteins

Abstract

Vibrational energy flow in biological macromolecules can be nicely studied by ultrafast pump-probe spectroscopy, given that suitable chromophors for injecting and tracking energy flow are present. Here we present a study on a new donor-acceptor pair consisting of two artificial aminoacids, with an azulene chromophore as a “donor”, that can be excited at 600 nm and injects vibrational energy into the peptide or protein investigated. An azide chromophore is serving as an “acceptor”, which can be monitored at 2100 cm-1 to track energy flow in the system. These labels combine a set of very favourable properties for the study of IVR in biological macromolecules. Co-translational incorporation of each of the labels into proteins has been demonstrated in the form of beta-(1-azulenyl)-L-alanine and L-azido-homo-alanine. To investigate the performance of the azulene-azide donor-acceptor pair, we designed a model peptid (Aaa-Tyr-Asn-Aha-Gly) inculding both chromophores and additional protein marker modes, such as tyrosine, asparagine and glycine providing the c-terminal carboxyl. We performed Vis-pump IR-probe experiments on the peptide, covering the range from 1200 cm-1 to 2120 cm-1. While in azulene-containing monomers, studied for comparison, the infrared signals reached their maximum within 2ps, we found for the peptide a pronounced correlation between the through-bond distance of a vibrating group from the azulene chromophore and the time until the IVR induced signal of this group becomes maximal. The signal of the azide band of our “acceptor” azido-homo-alanine is the dominating contribution at 9-10 ps. Even over a distance of four residues, it reaches a signal size comparable to the total amide I intensity of the peptide. In the light of the presented results the application of the azulene-azide donor-acceptor pair for IVR studies in proteins appears very promising.