We investigate protein structure and function using biochemical and biophysical methodologies with emphasis on taking advantage of the intrinsic tryptophan fluorescence and phosphorescence properties of proteins. Proteins investigated include the mannitol transporter, EIImtl from E. coli, domain 1 of transhydrogenase from Rhodospirillum rubrum and LysM domains of N-acetylglucosamidase from Lactococcus lactis. Single Trp-containing mutants of these proteins have been created and in most studies, these constructs were labeled with a Trp analog using Trp auxotroph expression hosts. In this way more information about the protein could be obtained than using Trp as spectroscopic probe. For example, an interesting property of 7-azatryptophan and 5-hydroxytryptophan is their red-shifted absorption spectrum relative to Trp (Figure 1). Therefore, proteins containing these analogs can be specifically excited >310 nm in the presence of Trp containing proteins.
Fig. 1. Absorption spectra of tryptophan and tryptophan analogs.
Highlights of our research include:
Development of an expression system for the efficient incorporation of an expended set of Trp analogs
- coli auxotrophs are most often used for the residue-specific incorporation of unnatural amino acids in proteins. As a result of the high fidelity of the translation system only pseudo-isosteric analogs of the canonical amino acids can be translated. For example, known Trp auxotrophs of E. coli can only translate half a dozen different Trp analogs with useful properties (1). We became aware of other Trp analogs with exciting (spectroscopic) properties and this triggered the search for new protein expression systems. This resulted in a Lactococcus lactis Trp auxotroph protein expression system, coexpressing the tryptophanyl tRNA synthetase (TrpRS) (2,3). This system is the most versatile Trp analog expression system known to date, as next to the Trp analogs known to be translated by E. coli, at least 6 more useful Trp analogs can be efficiently translated by this system. It includes β-(1-azulenyl)-l-alanine a blue colored Trp analog (4)(Figure 1).
Figure 2. Side chain structures of Trp analogs which are translated by Lactococcus lactis Trp auxotroph strain PA1002, coexpressing the tryptophanyl tRNA synthetase.
5-Fluorotryptophan decays monoexponentially when embedded in proteins
We discovered 5-fluorotryptophan (5-FTrp) most often decays monoexponentially when embedded in a protein structure while these mutants harbouring a Trp decays multiexponential (3-4 lifetimes) (5). In a collaborative study with the groups of prof Callis (Montana State University) and prof Buma (Amsterdam University) the impact of the 5-fluoro substitution on the fluorescence decay properties was studied in more detail (6). Theoretical and experimental work support the notion that photo-induced electron transfer from indole to the peptide backbone is essentially suppressed by 5-fluoro substitution. This process is held responsible for the presence of multiple lifetimes of Trp in proteins.
The monoexponential decay of 5-FTrp makes possible to extract much more information using fluorescence spectroscopy, in particular if 5-fluorotryptophan is used as donor in time-resolved fluorescence resonance energy transfer (RET) experiments. Comparison of the lifetimes in absence and presence of acceptor allows easy calculation of the distance between donor and acceptor (5).
Localization of the mannitol binding site in the dimeric mannitol transporter, EIImtl
5-fluorotryptophan (5-FTrp) was incorporated in a dozen single Trp mutants of EIImtl (7). This is a homodimeric sugar transporter, thus two 5-fluorotryptophan labels are present per functional oligomer. This homodimeric transporter harbours a single high affinity mannitol binding site and to localize this site using RET, mannitol was equipped with an diaziridine moiety (azi-mannitol)(Figure 3), the smallest known acceptor for Trp (analog) fluorescence (8). In Figure 3 the fluorescence decay is shown of 5-FTrp labeled mutant W30 in absence and presence of azi-mannitol. The amplitude (y-axis) of the monoexponential decaying W30 reduces 50% upon binding azi-mannitol, while the lifetime (slope of the decay) of the remaining fluorescence is not affected. This result shows azi-mannitol binds very close to one of the two 5-FTrp rsidues in W30, completely quenching its fluorescence, while the other 5-FTrp residue is too distant to become quenched by azi-mannitol. Thus two distances are obtained in this experiment.
Figure 3. Left: Structures of the donor (5F-Trp) and acceptor (azi-mannitol) and their emission/absorption spectra. Right: Fluorescence decay spectrum of 5-FTrp-labeled EIImtl mutant W30 in the absence (black) and presence (red) of a saturation about of azi-mannitol.
This RET study stands out among other protein RET studies because:
- a donor-acceptor couple was used imposing minimal native structure disruption
- ii) the donor-acceptor couple features one of the shortest Forster distances known (10Å), making this couple ideal to measure with high accuracy distances between 8-16 Å.
- the monoexponential decay of 5-FTrp makes possible to measure 2 distances simultaneously in a single experiment.
Detection of 1Lb emission of tryptophan in proteins
The absorption band of indole at approximately 280 nm comprises two overlapping transitions to the 1La and 1Lb excited states. Upon excitation, 1La emission is observed except when the indole is excited in the gas state or when dissolved in fluorinated hydrocarbon. We reported for the first time 1Lb emission can also be observed for Trp embedded in an extremely rigid and apolar proteineous environment (9,10). Several mutants of domain 1 (D1) of the transhydrogenase protein from Rhodospirillum rubrum, show 1Lb emission and also feature the bluest emission maximum reported for Trp in a protein (λmax = 304 nm) (Figure 4). Quantum mechanical/ molecular mechanical calculations indicated the 1La state is minimally stabilized by the rigid protein matrix, surrounding the Trp, making 1Lb the emitting state (9).
Figure 4. Normalized emission spectra of apo-azurin (black) (λmax= 306.5 nm), Wt dI (red) (λmax= 308.0 nm) and dI.M97V (blue) (λmax= 303.5 nm) in buffer, pH=7.4, at room temperature. Only mutant dI.M97V shows 1Lb emission.
- Broos, J (2014) Biosynthetic incorporation of tryptophan analogs in proteins Methods Mol. Biol. 1076, 359-370
- Petrovic, D M, Leenhouts, K, van Roosmalen, M L, and Broos, J (2013) An expression system for the efficient incorporation of an expanded set of tryptophan analogues Amino Acids 44, 1329-1336
- Shao, J, Marcondes, M F, Oliveira, V, and Broos, J (2016) Development of chemically defined media to express Trp-analog-labeled proteins in a Lactococcus lactis Trp auxotroph J. Mol. Microbiol. Biotechnol. 26, 269-276
- Shao, J, Korendovych, I V, and Broos, J (2015) Biosynthetic incorporation of the azulene moiety in proteins with high efficiency Amino Acids 47, 213-216
- Broos, J, Maddalena, F, and Hesp, B H (2004) In vivo synthesized proteins with monoexponential fluorescence decay kinetics J. Am. Chem. Soc. 126, 22-23
- Liu, T Q, Callis, P R, Hesp, B H, de Groot, M, Buma, W J, and Broos, J (2005) Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins J. Am. Chem. Soc. 127, 4104-4113
- Opacic, M, Vos, E P, Hesp, B H, and Broos, J (2010) Localization of the substrate binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli J. Biol. Chem. 285, 25324-25331
- Broos, J, Pas, H H, and Robillard, G T (2002) The smallest resonance energy transfer acceptor for tryptophan J. Am. Chem. Soc. 124, 6812-6813
- Broos, J, Tveen-Jensen, K, de Waal, E, Hesp, B H, Jackson, J B, Canters, G W, and Callis, P R (2007) The emitting state of tryptophan in proteins with highly blue-shifted fluorescence Angew. Chem. , Int. Ed. 46, 5137-5139
- Tveen-Jensen K., Strambini, G B, Gonnelli, M, Broos, J, and Jackson, J B (2008) Mutations in transhydrogenase change the fluorescence emission state of trp-72 from 1La to 1Lb Biophys. J. 95, 3419-3428