Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae

Article abstract of DOI:10.1021/ja904896s

(Graph Presented) Here, we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be genetically incorporated into proteins in yeast with excellent selectivity and efficiency by means of an orthogonal tR

Full text of DOI:10.1021/ja904896s

Published on Web 08/24/2009
Genetic Incorporation of a Small, Environmentally Sensitive, Fluorescent
Probe into Proteins in Saccharomyces cerevisiae
Hyun Soo Lee, Jiantao Guo, Edward A. Lemke, Romerson D. Dimla, and Peter G. Schultz*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
Received June 15, 2009; E-mail: schultz@scripps.edu
The introduction of fluorescent probes into proteins provides a
powerful tool to study protein structure and function either in vitro
or in vivo.1 One particularly useful method involves genetically
fusing the protein of interest to fluorescent proteins.2 However, the
size of naturally occurring fluorescent proteins (>20 kDa) and the
typical requirement for C- or N-terminal fusions limit their utility
as a local probe of structure and in some cases can perturb the
structure or function of a protein. A variety of other methods have
been developed to introduce fluorescent probes into protein in vitro
and in vivo including the selective chemical3 or enzymatic4
modification of specific recognition motifs, the use of intein-based
semisynthetic methods,5 and in vitro protein translation6 with
chemically aminoacylated tRNAs. Although very useful, each
approach has its own limitations including protein size and yields,
modifications to the native protein sequence, or reagents/protocols
that are not compatible with live cells. To augment and extend these
methods, we have attempted to genetically encode small, fluorescent
amino acids directly in living cells.7 In this study, we report the
site-specific incorporation of an environmentally sensitive, fluo-
rescent amino acid into proteins in response to the amber nonsense
codon (TAG) with good yield and high fidelity in yeast. We
demonstrate that the method can be used as a local probe of
conformational changes in protein structure induced by ligand
binding.
6-Propionyl-2-(N,N-dimethyl)aminonaphthalene, prodan,8 is a
highly environmentally sensitive fluorophore that is widely used
in biochemistry and cell biology.9 To genetically encode the amino
acid analogue of prodan, 3-(6-acetylnaphthalen-2-ylamino)-2-
aminopropanoic acid (Anap, 1), it was synthesized in six steps
starting with 1-(6-hydroxynaphthalen-2-yl)ethanone (Figure S1,
Supporting Information). The extinction coefficient (17 500 cm-1
M-1) and quantum yield (0.48, Figure S2, Supporting Information)
of Anap in EtOH (360 nm) are comparable to those of prodan
(18 400 cm-1M-1 and 0.71).8,10 The fluorescence spectra in various
Figure 1. (a) Chemical structures of Anap (1) and Nap (2). (b) Fluorescence
spectra of 1 in various solvents; samples were excited at 350 nm.
(M40, L41, Y499, Y527, and H537) in the leucine-binding site.7a,12
The library was then subjected to rounds of alternating positive
and negative selections as previously described,14 but unfortunately
no mutant LeuRS with the desired selectivity was isolated. We next
attempted a stepwise strategy15 in which the same selection scheme
was used first to evolve an aminoacyl-tRNA synthetase specific
for the Anap analogue, 3-(naphthalen-2-ylamino)-2-aminopropanoic
acid (Nap, 2). This amino acid lacks the acyl substituent of Anap
and therefore requires a lower degree of hydrogen bonding
complementarity in the amino acid binding site of LeuRS. Five
rounds of selection generated 92 clones that survived only in the
presence of Nap in uracil dropout medium; DNA sequencing of
solvents also exhibit sensitivity to solvent polarity similar to other
max
prodan probes with a significant shift in emission maximum (λ
)
em
max
max
on transitioning from water (λ ) 490 nm) to ethyl acetate (λ
em
em
) 420 nm) (Figure 1). Compared with dansyl- or coumarin-
containing amino acids, Anap has enhanced environmental sensi-
tivity with comparable or increased brightness.9c,11
To genetically encode this amino acid in Saccharomyces cer-
eVisiae, an orthogonal Escherichia coli amber suppressor tRNA/
Table 1. Selected Anap-Specific LeuRS Mutants
leucyl-tRNA synthetase (tRNA /LeuRS) pair12 was used on the
Leu
Leu Met Leu Tyr Tyr Tyr His Leu Phe Ala
40
CUA
LeuRS
frequency 38
41 499 500 527 537 538 541 560
basis of previous experiments7a,13 which showed that LeuRS
Anap-1
10/10 Phe Gly Pro Gly Tyr Ala Thr Cys Ser Val
5/10 Phe Gly Pro Val His Ala Glu Ser Cys Val
3/10 Phe Gly Pro Ile His Ala Glu Ser Cys Val
2/10 Phe Gly Pro Val Leu Ala Glu Ser Cys Val
mutants can accommodate unnatural amino acids with large
Anap-2A
Aanp-2B
Anap-2C
hydrophobic side chains. To alter the specificity of LeuRS so that
Leu
it aminoacylates tRNA
with Anap and no endogenous amino
CUA
acids, a LeuRS library was constructed by randomizing five residues
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Figure 3. Fluorescence measurements of QBP-Anap with different
concentrations of Gln. Protein samples (10 nM) in D-PBS were excited at
350 nm. Fluorescence spectra were recorded after adding Gln in the same
buffer to the final indicated concentrations without further incubation. (a)
Fluorescence spectra. (b) Fluorescence intensity at 430 nm.
Figure 2. Expression of hSOD with an amber codon at position 33 in the
Leu
that survived only in the presence of Anap in uracil dropout
medium; DNA sequencing of 10 clones revealed only one unique
mutant (Anap-1, Table 1). This mutant LeuRS was used to express
hSOD-33TAG to test its ability to selectively incorporate Anap into
proteins. SDS/PAGE analysis and MALDI-TOF MS showed that
the incorporation efficiency of Anap into hSOD-33TAG by the
mutant LeuRS was poor and that the mutant LeuRS incorporated
natural amino acids (Leu or Ile) to a significant degree (data not
shown). To improve the efficiency and selectivity of Anap-1,
another library of LeuRS mutants was generated by randomizing
eight residues (L38, M40, Y499, Y500, H537, L538, F541, and
A560) (Lib. 2, Table S2, Supporting Information) based on the
sequences of the LeuRS mutants from the previous selections. After
five rounds of selection, three unique mutants with the desired
phenotype were identified (Table 1), and the ability of one of these
clones (Anap-2C: F38, G40, P41, V499, L500, A527, G537, S538,
C541, and V560) to incorporate Anap into hSOD-33TAG was
tested. SDS/PAGE analysis and ESI-MS showed that the mutant
LeuRS incorporated Anap into the protein with good efficiency (1.2
mg/L in shake flasks) and selectivity (Figure 2).
presence of the tRNA /Anap-2C pair and 0.4 mM Anap. (a) SDS-PAGE
CUA
analysis by GelCode Blue straining (top) and fluorescence (bottom). Lane
Leu
1, protein marker; Lane 2, hSOD expressed in the presence of the tRNA
/
CUA
Anap-2C pair and 0.4 mM Anap; Lane 3, hSOD expressed in the presence
Leu
of the tRNA /Anap-2C pair, but in the absence of Anap. (b) ESI-MS
CUA
analysis. The inset shows the deconvoluted spectrum: calculated 16 738,
observed 16 736.
10 clones revealed three unique mutants (Table S1, Supporting
Information). The ability of one of these clones, Nap-1 (G40, P41,
G499, A527, T537), to selectively incorporate Nap into proteins
was tested by suppression of an amber mutation in C-terminal His6-
tagged human superoxide dismutase (hSOD-33TAG) in the presence
of Nap-1 and 1 mM Nap. SDS/PAGE analysis and MALDI-TOF
MS showed selective incorporation of Nap with little detectable
incorporation of endogenous yeast amino acids (Figure S3, Sup-
porting Information). The mutation of the Met, Leu, and Tyr
residues of wild-type LeuRS to small hydrophobic residues in Nap-1
is consistent with the generation of a larger binding pocket to
accommodate the naphthyl side chain of 2.
Next, a LeuRS library was generated based on the sequences of
the LeuRS mutants evolved for Nap and the crystal structure of
Thermus thermophilus LeuRS (which is homologous to E. coli
LeuRS).16 Residues 40, 41, 499, 527, and 537 were fixed as Gly,
Pro, Gly, Ala, and Thr, respectively, and five additional residues
(L38, Y500, L538, F541, and A560) in the leucine- binding site
were randomized (Lib. 1, Table S2, Supporting Information). Five
rounds of selection for Anap-specific synthetases afforded 61 clones
We next asked whether our ability to site-specifically incorporate
Anap into proteins could be used to measure ligand-induced local
conformational changes in proteins directly without the need for a
FRET pair, using glutamine-binding protein (QBP) from E. coli.
QBP is a monomeric protein composed of 224 amino acid residues
that is responsible for the first step in the active transport of
L-glutamine across the cytoplasmic membrane.17 QBP consists of
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C O M M U N I C A T I O N S
two similar globular domains which are linked by two peptide
hinges.17 The X-ray crystal structure of QBP shows that the deep
cleft formed between the two domains contains the glutamine
binding site, and glutamine binding causes a significant conforma-
tional change.17 To incorporate Anap into QBP, an amber nonsense
codon was substituted for Asn160,18 which lies in the cleft between
the two domains. Expression and purification of the amber mutant
Acknowledgment. We thank Dr. Lei Wang for providing
plasmids for protein expression. We are grateful to the NIH
GM62159 and the Skaggs Institute for Chemical Biology for support
of this work.
Supporting Information Available: Materials and methods. This
material is available free of charge via the Internet at http://pubs.acs.org.
of QBP containing a C-terminal His6 tag (QBP-160TAG) was
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