PhD ceremony Mr. D. Pétrovic: Efficient biosynthetic incorporation of tryptophan analogues, and spectroscopic characterization of tryptophan-analogue labelled LysM proteins

PhD ceremony: Mr. D. Pétrovic

Dissertation: Efficient biosynthetic incorporation of tryptophan analogues, and spectroscopic characterization of tryptophan-analogue labelled LysM proteins

Promotor(s): prof. B.W. Dijkstra

Faculty: Mathematics and Natural Sciences

In the last few decades, fluorescence spectroscopy has become a primary research tool in the life sciences. Trp has attractive spectroscopic properties to study protein structure and dynamics, like a high sensitivity of the spectral energy and quantum yield for a change in microenvironment. In practice, these features can only be optimally exploited if purified proteins are used containing only one or two Trp residues.

One approach to overcome this limitation is replacing the Trp of interest by a Trp analog. These Trp analogue labeled proteins are also known as spectrally enhanced alloproteins. One of the goals of this PhD work was to explore the use of these proteins as molecular recognition element in a biosensor. Moreover, new routes are presented for the production of alloproteins.

A sensitive spectroscopic method allowing the direct monitoring of the binding event has not been reported. In chapter 2 a fluorescence method is presented to directly measure the interaction of a LysM domain with peptidoglycan. A Trp analog fluorescence spectroscopy based method is presented needing only a few µg of LysM.

In Chapter 3 a Lactococcus lactis Trp auxotroph expression system is presented able to biosynthetically incorporate tryptophan analogs with bulky substituents.

In chapter 4 convincing experimental data is presented about the emitting state of 5HI, 5HW, and of 5HW incorporated in 4 proteins. ¹L_b is the emitting state in all these samples except in one protein, which shows dual emission from both the ¹L_a and ¹L_b states.