Download small heat shock protein activity is regulated by

Kinetic analysis of the leucyl/phenylalanyl-tRNAprotein transferase with acceptor peptides
possessing different N-terminal penultimate
residues
Jun Kawaguchi a, Kumino Maejima b, Hiroyuki Kuroiwa b, Masumi Taki a,b,*
Supplementary material
Contents:
1. Synthesis of O-(2-fluoroethyl)-L-tyrosine
2. Acid urea PAGE analysis of aminoacyl-tRNA
3. Quantitative measurement of initial reaction rate by MALDI-TOF-MS
4. Lineweaver-Burk plot of several acceptor peptides with different penultimate
residues
5. Supporting references
1
1. Synthesis of O-(2-fluoroethyl)-L-tyrosine
Overall scheme for synthesis of O-(2-fluoroethyl)-L-tyrosine is shown below.
O
S Cl
O
HO
O
F
CH2Cl2, pyridine
4-dimethylaminopyridine
S
O
F
O
O
O
HN
HO
O
O
O
O
F
MeCN, CH3ONa
HN
O
O
OH
1) NaOH
2) TFA
O
F
O
NH2
O

2
1-1. Fluoroethyl tosylate
O
HO
F
+
S Cl
O
r. t.
O
CH2Cl2, pyridine
4-dimethylaminopyridine
S
O
O
F
To a chilled solution of tosyl chloride (6.9 g, 36 mmol) and 2-fluoroethanol (1.9
g, 30 mmol) in dry dichloromethane (30 mL), 4-dimethylaminopyridine (0.10 g, 0.85
mmol) in dry pyridine (10 mL) was added dropwise for 15 minutes under argon
atmosphere. The mixture was stirred 63 hr initially at 0o C and then at room temperature
in the dark. The mixture was mixed with chilled 5% aqueous HCl and extracted with
dichloromethane. The extract was washed twice with 5% aqueous HCl, twice with
saturated brine, dried over magnesium sulfate, and concentrated under reduced pressure.
Purification by silica-gel column chromatography (hexane/AcOEt = 3:1) gave 1.6 g (25%
yield) of desired product as colorless oil.
Fluoroethyl tosylate: TLC Rf = 0.40 (hexane/AcOEt = 3:1); 1H NMR (300 MHz, CDCl3)
spectrum is shown below.
3
Figure S1. 1H NMR spectrum of fluoroethyl tosylate.
1-2. N-Boc-fluoroethyl-L-tyrosine methyl ester
O
O
reflux
O
O
S
O
O
+
F
HO
HN
MeCN, CH3ONa F
O
O
O
HN
O
O
O
Fluoroethyl tosylate (0.76 g, 3.5 mmol), N-Boc-L-tyrosine methyl ester (1.0 g, 3.5 mmol),
and sodium methoxide (0.19 g, 3.5 mmol) were mixed in dry acetonitrile (6 mL),
refluxed for 12 hr, and brought to room temperature. The crude reaction product was
mixed with saturated aqueous solution of NH4Cl, and extracted with ethyl acetate. The
extract was dried over magnesium sulfate, and concentrated under reduced pressure to
4
afford amber-colored oil.
Purification by silica-gel column chromatography
(hexane/AcOEt = 3:1) gave the mixture of desired N-Boc-fluoroethyl-L-tyrosine methyl
ester and unfavorable fluoroethyl tosylate as white turbid oil. As the impurity (fluoroethyl
tosylate) could be removed at the next step, the mixture was used for the next reaction
without further purification.
N-Boc-fluoroethyl-L-tyrosine methyl ester: TLC Rf = 0.30 (hexane/AcOEt = 3:1); 1H
NMR (CDCl3) spectrum is shown below.
Figure S2. 1H NMR spectrum of N-Boc-fluoroethyl-L-tyrosine methyl ester.
5
1-3. O-(2-fluoroethyl)-L-tyrosine
OH
O
O
F
O
HN
O
heat
O
MeOH, NaOHaq F
O
HN
O
O
O
OH
O
TFA
F
O
NH2
To a solution of N-Boc-fluoroethyl-L-tyrosine methyl ester (74 mg) in MeOH
(0.85 mL), 85 M of 2 M NaOH in water was added dropwise. The mixture was stirred
for 2.5 hr at 70o C and then the solvent was evaporated. The mixture was acidified to pH
1 with aqueous HCl and extracted three times with each 0.4 mL of CHCl3. The extract
was dried over sodium sulfate, and concentrated under reduced pressure to afford desired
N-Boc-fluoroethyl-L-tyrosine. The deprotection of Boc group of N-Boc-fluoroethyl-Ltyrosine with trifluoroacetic acid (TFA) was carried out by following the same procedure
as previously described[1], and the obtained white solid of O-(2-fluoroethyl)-L-tyrosine
(19 mg) was used for the chemoenzymatic reaction without further purification.
1
H NMR (D2O) spectrum of O-(2-fluoroethyl)-L-tyrosine is shown below.
6
Figure S3. 1H NMR spectrum of O-(2-fluoroethyl)-L-tyrosine.
2. Acid urea PAGE analysis of aminoacyl-tRNA
During the time period of kinetic analysis, molar concentration of the aminoacyl-tRNA
was kept constant (8.2 M). This was confirmed by acid urea polyacrylamide gel
electrophoresis (acid urea PAGE), as aminoacyl-tRNA is not hydrolyzed around pH 5.
The acid urea PAGE was performed according to the reported method[2], and result was
shown in Fig. S4. Under the experimental condition described in the main text, all the
tRNA molecules were aminoacylated because mutant FRS regenerate aminoacylated
tRNA from hydrolyzed one.
7
Figure S4. Acid urea PAGE analysis of aminoacyl-tRNA. After electrophoresis, tRNA
was stained with SYBR Gold, and fluorescence imaging in the gel under excitation at 488
nm was performed by using a FMBIO III-SC01 (Hitachi, Japan) with a band-pass filter
(555 BP20) for the detection. Natural and non-natural amino acids, phenylalanine (Phe)
and O-(2-fluoroethyl)-L-tyrosine, respectively, was transferred to 3’ end of all tRNA
molecules in the presence of mutant FRS. Molecular weight of aminoacyl-tRNA is
slightly higher than that of deacylated tRNA.
8
3. Quantitative measurement of the initial reaction rate by MALDI-TOF-MS
The initial reaction rate of the L/F-transferase-mediated amino acid transfer was
measured by following a procedure of quantitative MALDI-TOF-MS analysis reported
previously[3, 4]. An example for determining the initial reaction rate is presented in Fig.
S5.
Figure S5. An example of MALDI-TOF-MS spectra of sequential time points of L/Ftransferase mediated non-natural amino acid transfer reaction. The reaction rates of all
the other light acceptor peptides possessing different penultimate residues were measured
in the same way. (a) In this case, light acceptor peptide with threonine as the penultimate
residue (KTC*-acdAla, m/z 642) was used. Note that disappearance of light peptide (m/z
642) and appearance of product (feTyr-KTC*-acdAla, m/z 851) as the reaction proceeds
9
from 3 to 60 min. The molar concentration of the light peptide was quantified by the ratio
intensities of the light one to the heavy standard one (m/z 647). (b) Graphical analysis of
disappearance of light peptide. Initial reaction rate (V0) was estimated from time course
of the molar concentration of remaining light peptide.
4. Lineweaver-Burk plot of several acceptor peptides
Kinetic parameters for phenylalanine (Phe) transfer from Phe-tRNA to each
acceptor peptide catalyzed by L/F-transferase were estimated from Lineweaver-Burk
plot. A straight line is formed by plotting the inverse initial reaction rate (1/V0) as a
function of the inverse of the acceptor peptide concentration (1/[S]). The 1/Vmax and 1/Km values were determined from y- and x-intercepts, respectively. A detailed example
for determining the kinetic parameter is presented in Fig. S6.
10
Figure S6. Lineweaver-Burk plot for an acceptor peptide whose sequence is
RGPC*RAFI. Cysteine (C*) was alkylated with bromomethane for quantitative MALDITOF-MS analysis. The standard deviation was represented as R-squared (R2) value.
Kinetic parameters for Phe transfer to each acceptor peptide with different
penultimate residue were also estimated and shown in Fig. S7.
Figure S7. Lineweaver-Burk plot for each acceptor peptide possessing different
penultimate residue. In these cases, cysteine was not alkylated and the plot was taken
from HPLC analysis.
11
5. Supporting references:
[1] M. Taki, T. Hohsaka, H. Murakami, K. Taira, and M. Sisido, A non-natural amino
acid for efficient incorporation into proteins as a sensitive fluorescent probe. FEBS Lett
507 (2001) 35-38.
[2] C. Kohrer, and U.L. Rajbhandary, The many applications of acid urea polyacrylamide
gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases. Methods 44
(2008) 129-138.
[3] H.A. Ebhardt, Z. Xu, A.W. Fung, and R.P. Fahlman, Quantification of the posttranslational addition of amino acids to proteins by MALDI-TOF mass spectrometry.
Anal Chem 81 (2009) 1937-1943.
[4] A.W. Fung, H.A. Ebhardt, H. Abeysundara, J. Moore, Z. Xu, and R.P. Fahlman, An
alternative mechanism for the catalysis of peptide bond formation by L/F transferase:
substrate binding and orientation. J Mol Biol 409 (2011) 617-629.
12