2 research outputs found
Fluorescent Biphenyl Derivatives of Phenylalanine Suitable for Protein Modification
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 (<i>ec</i>DHFR) 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 <i>ec</i>DHFR. All phenylalanine derivatives were incorporated
with good efficiency into position 16 of <i>ec</i>DHFR 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 <i>ec</i>DHFR, 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
Fluorescent Biphenyl Derivatives of Phenylalanine Suitable for Protein Modification
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 (<i>ec</i>DHFR) 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 <i>ec</i>DHFR. All phenylalanine derivatives were incorporated
with good efficiency into position 16 of <i>ec</i>DHFR 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 <i>ec</i>DHFR, 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