ABSTRACT
For novel insights into the pathogenicity of
Candida albicans
, studies on molecular interactions of central virulence factors are crucial. Since methods for the analysis of direct molecular interactions of proteins
in vivo
are scarce, we expanded the genetic code of
C. albicans
with the synthetic photo-cross-linking amino acid
p
-azido-
l
-phenylalanine (AzF). Interacting molecules in close proximity of this unnatural amino acid can be covalently linked by UV-induced photo-cross-link, which makes unknown interacting molecules available for downstream identification. Therefore, we applied an aminoacyl-tRNA synthetase and a suppressor tRNA pair (
Ec
TyrtRNA
CUA
) derived from
Escherichia coli
, which was previously reported to be orthogonal in
Saccharomyces cerevisiae
. We further optimized the aminoacyl-tRNA synthetase for AzF (AzF-RS) and
Ec
TyrtRNA
CUA
for
C. albicans
and identified one AzF-RS with highest charging efficiency. Accordingly, incorporation of AzF into selected model proteins such as Tsa1p or Tup1p could be considerably enhanced. Immunologic detection of C-terminally tagged Tsa1p and Tup1p upon UV irradiation in a strain background containing suppressor tRNA and optimized AzF-RS revealed not only the mutant monomeric forms of these proteins but also higher-molecular-weight complexes, strictly depending on the specific position of incorporated AzF and UV excitation. By Western blotting and tandem mass spectrometry, we could identify these higher-molecular-weight complexes as homodimers consisting of one mutant monomer and a differently tagged, wild-type version of Tsa1p or Tup1p, respectively, demonstrating that expanding the genetic code of
C. albicans
with the unnatural photo-cross-linker amino acid AzF and applying it for
in vivo
binary protein interaction analyses is feasible.
Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code
