Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification

Abstract

In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational flexibility about the biphenyl bonds.

Figure 1

Biphenyl-phenylalanine (A – D) and L-(7-hydroxycoumarin-4-yl)ethylglycine (E) amino acids prepared and incorporated into dihydrofolate reductase.

Figure 2

Autoradiogram of a 15% SDS-polyacrylamide gel (100 V, 2 h) illustrating the incorporation of biphenyl-phenylalanine derivatives into positions 16 (upper panel) and 49 (lower panel) of DHFR. Lane 1, wild-type DHFR expression; lane 2, modified DHFR DNA in the presence of abbreviated suppressor tRNA_CUA-C_OH; lane 3, incorporation of amino acid A; lane 4, incorporation of amino acid B; lane 5, incorporation of amino acid C; lane 6, incorporation of amino acid D. Phosphorimager analysis was performed using an Amersham Biosciences Storm 820 equipped with ImageQuant version 5.2 software from Molecular Dynamics.

Figure 3

MALDI-MS of tryptic fragments of wild-type (panel A) and modified DHFR containing biphenyl-phenylalanine C at position 49 (panel B) (*: calculated value in Da; ^#: cysteine was alkylated with 2-iodoacetamide; bip: biphenyl-phenylalanine derivative C). Figure 4.

Figure 4

Fluorescence emission spectra of DHFRs containing amino acid B at positions 16 (black trace) and 49 (green trace).

Figure 5

Fluorescence of modified DHFRs (0.3 µM) containing L-(7-hydroxycoumarin-4-yl)ethylglycine (E) at position 17 and biphenyl-phenylalanine A (red curve), B (green curve), C (blue curve) or D (purple curve) at position 115. The spectra were recorded at pH 8.0 with λ_ex = 280 nm.

Scheme 1

Synthetic Route Employed for the Preparation of Biphenyl-phenylalanyl-pdCpA 6

Scheme 2

Strategy Employed for Incorporation of Biphenyl-phenylalanine C into DHFR at Position 115.