1 Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA 2 Department of Animal and Dairy Sciences, Auburn University, Auburn, AL 36849, USA
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Abstract |
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Keywords: calcium-binding protein/CD2/design/fluorescence energy transfer/Tb(III) binding
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Introduction |
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Previous work with the cell adhesion protein CD2-D1 has demonstrated that this protein is an excellent choice as a host system for engineering a calcium-binding site (Yang, J.J. et al., 2000a,b
). CD2-D1 is a small ß-sheet protein (99 amino acids) with a common IgG fold (Figure 1
). Large conformational changes are not observed in the pH range 110 (Yang,J.J et al., 2000a
). Moreover, it produces a high expression yield in BL-21. CD2-D1 can also be reversibly refolded after chemical (guanidinium.HCl) (Tanford, 1968
) and thermal denaturation (Yang,J.J et al., 2000b
). NMR assignments (Davis and van der Merwe, 1996
) and X-ray structures (Jones et al., 1992
) are available. CD2-D1 has been shown to accommodate a calcium-binding pocket (Ye et al., 2001
). CD2-D1 has been shown to withstand up to
40 separate mutations without changing conformation (Arulanandam et al., 1993
).
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Materials and methods |
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Computational design of calcium-binding site
The protein is designed using established methods based on the local geometry of the calcium-binding site. CD2-D1 (1hng) was used as the host protein. The designed sites were screened based on their geometric deviations, solvent accessibility, side chain clashes and charge numbers.
Protein expression and purification
The designed DEEEE protein was made using site-directed mutagenesis. Automated DNA sequencing confirmed that the mutant had been successfully made.
LB broth with ampicillin was used for growth and expression of the GST-fusion CD2 variant. The variant was grown until an OD600 of 0.8 and induced with 0.1 mM isopropyl-ß-D-thiogalactoside (IPTG). Purification was completed using affinity chromatography with GS4B beads (AmershamPharmacia) and the GST-tag was cleaved on the column with 40 units/ml of PreScission protease (Amersham-Pharmacia) to obtain pure CD2 variant.
Both electrospray ionization (ESI) and the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were used to verify the correct molecular mass and purity of the DEEEE protein. SDSPAGE analysis was also used to measure the purity.
The protein was dialyzed against 10 mM TrisHCl, pH 7.4. The concentration was measured using a UV-1601 spectrophotometer (Shimadzu) interfaced to a PC at 280 nm ( = 11 700) (Driscoll et al., 1991
).
Fluorescence resonance energy transfer (FRET)
A PTI fluorimeter was used with an excitation slit width of 6 nm and an emission slit width of 25 nm. A glass filter was placed in front of the exit slit of the emission to filter out the secondary Raleigh reflection. The excitation wavelength was set at 283 nm and the emission scan was from 500 to 600 nm, with an expected emission peak at 545 nm. An average of two scans was taken. A sample of 30 µM Tb(III) and a stock solution of 20 µM DEEEE with 30 µM Tb(III) were prepared using 10 mM Tris, pH 7.4. FRET with increasing protein concentration was measured by adding 100 µl aliquots of the stock solution directly to 30 µM Tb(III) with an equilibration time of 15 min. The emission fluorescence was measured. This procedure was continued until a final concentration of 8.0 µM protein was reached.
Two samples were prepared for competition binding studies, one containing 30 µM Tb(III) and 8 µM DEEEE with 100 µM La(III) and the other 30 µM Tb(III) and 8 µM DEEEE with 10 mM Ca(II). The samples were equilibrated at room temperature for 2 h and measured by averaging two scans.
Once the intensity enhancement by protein had been determined at 545 nm, a titration with increasing terbium was performed. Samples of DEEEE with concentrations of 46, µM and samples of 1 mM Tb(III) with 4 and 6 µM DEEEE were prepared. The same parameters were used as indicated previously for analysis by energy transfer.
Data analysis
The fractional change versus the Tb(III) concentration was plotted with normalized fluorescence intensity at 545 nm. This was done by subtracting the intensity of free terbium from the sample of protein with terbium with baseline correction. The baseline shift was corrected by integrating areas between 527568 nm. The obtained intensities of Tb(III) with protein were further subtracted from the intensity of free Tb(III) at the same concentrations.
The dissociation constant Kd was calculated using the following equation:
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where F is the fractional change of Tb(III) fluorescence enhancement at 545 nm, [P]T is the total protein concentration and [M]T is the total metal concentration (Yang,W. et al., 2000b).
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Results and discussion |
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Using the established structural parameters from our previous studies on natural calcium-binding proteins, a single calcium-binding site with the common geometry of pentagonal bipyramid was designed in the host protein CD2 using the computer algorithm Dezymer (Hellinga and Richards, 1991; Yang,W. et al., 2000a
., 2001). Figure 1
shows a schematic representation of CD2-D1 containing the designed site (DEEEE), with the mutations G61E as the bidentate ligand and I18D, F21E, V80E and I88E as monodentate ligands in the loop regions and in two ß-strands. One ligand position in the geometry remains open to permit water to act as a bridge and to avoid molecular crowding, which is similar to natural calcium-binding proteins (Falke et al., 1994
). The location of the designed site should allow for water accessibility and a way to monitor metal induced conformational change. Further, a net charge of 5 in the coordination sphere was chosen to ensure strong metal-binding affinity especially to lanthanide ions with similar ionic radii as calcium but with higher positive charge (Horrocks, 1993
).
Metal-binding ability monitored by fluorescence resonance energy transfer
Tb(III) has been widely used to probe calcium binding because of its similar ionic radius and coordination properties to those of calcium (Horrocks, 1993; Drake et al., 1997
). Terbium directly competes for calcium-binding sites in natural calcium-binding proteins, such as calmodulin and galactose binding protein (Kilhoffer et al., 1980
; Wang et al., 1982
; Falke et al., 1994
). Moreover, terbium is intrinsically fluorescent, with a broad excitation spectrum and an emission maximum at
545 nm. The excitation spectrum of terbium overlaps the emission spectrum of tryptophan and tyrosine, which allows for monitoring of terbium binding to CD2-D1 by energy transfer. As shown in Figure 1
, fluorescence energy transfer was used since Tyr81 and the buried Trp32 of DEEEE are
8.5 and
8.4 Å away from the binding pocket, respectively. The enhancement of the emission peak at 545 nm increases with the addition of protein to constant terbium, which suggests that terbium binds to the protein (Figure 2a
). Furthermore, this enhancement is only observed with the CD2 variant with designed DEEEE site. Wild-type CD2-D1 does not show this enhancement although it has the same four aromatic residues, Trp7 and -32 and Tyr76 and -81 (Figure 2b
).
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The fractional change versus the Tb(III) concentration was plotted by normalizing the fluorescence intensity at 545 nm and the baseline was corrected by using integrated areas between 527568 nm. (Figure 2c). This was completed by subtracting the intensities at 525 and 565 nm from that at 545 nm. The Tb(III) titration curve can be fitted to a 1:1 binding model as expected. Kd for both terbium titrations was calculated and the average was 21 ± 3 µM. This work is very exciting since the metal binding ability of our designed protein is about 15-fold stronger than that for the natural calcium-binding proteins with a similar Greek Key fold. Rajini et al. have reported that the Kd of
-crystallin for terbium is
300 µM (Rajini et al., 2001
). Now we can use this design approach to elucidate key determinants for calcium-binding affinity.
To test further the binding of terbium to the binding site, a competition of excess lanthanum (100 µM) and calcium (10 mM) with 30 µM terbium was performed. According to the published method for natural calcium-binding proteins such as calmodulin (Wang et al., 1982) and galactose binding protein (Drake et al., 1996
), excess lanthanum and calcium were used to examine whether the calcium or lanthanum compete for the same binding pocket as terbium. A decrease in the fluorescence intensity at 545 nm for DEEEE was observed in the presence of both lanthanum and calcium (Figure 3
), which suggests that these metals compete for the same binding pocket as terbium.
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Acknowledgements
We thank Dr Hillary Godwin of Northwestern University for the pGEX-PKT vector, Dr Homme Hellinga of Duke University Medical Center for the program Dezymer and Sarah Shealy for mass spectrometric analyses. We appreciate helpful discussions with Dr Robert Kretsinger, Lisa Jones, Timethia Bonner, Leanne Isley, April Ellis, Curt Coman and other members of Dr Jenny J.Yang's research group. This work was supported in part by GSU start-up funds, QIF, Research Initiation, Mentoring Grant and in part by the grants from the National Science Foundation (NSF) MCB-0092486 and NIH GM 62999-1 for J.J.Y.
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Notes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berridge,M.J., Bootman,M.D. and Lipp,P. (1998) Nature, 395, 645648.[CrossRef][ISI][Medline]
Davis,S.J. and van der Merwe,P.A. (1996) Science, 273, 12411242.[ISI][Medline]
DeGrado,W.F., Summa,C.M., Pavone,V., Nastri,F. and Lombardi,A. (1999) Annu. Rev. Biochem., 68, 779819.[CrossRef][ISI][Medline]
Downing,A.K., Driscoll,P.C., Harvey,T.S., Dudgeon,T.J., Smith,B.O., Baron,M. and Campbell,I.D. (1992) J. Mol. Biol., 225, 821833.[ISI][Medline]
Drake,S.K., Lee,K.L. and Falke,J.J. (1996) Biochemistry, 35, 66976705.[CrossRef][ISI][Medline]
Drake,S.K., Zimmer,M.A., Miller,C.L. and Falke,J.J. (1997) Biochemistry, 36, 99179926.[CrossRef][ISI][Medline]
Driscoll,P.C., Cyster,J.G., Campbell,I.D. and Williams,A.F. (1991) Nature, 353, 762765.[CrossRef][ISI][Medline]
Falke,J.J., Drake,S.K., Hazard,A.L. and Peersen,O.B. (1994) Q. Rev. Biophys., 27, 219290.[ISI][Medline]
Hellinga,H.W. and Richards,F.M. (1991) J. Mol. Biol., 222, 763785.[ISI][Medline]
Horrocks,W.D.,Jr. (1993) Methods Enzymol., 226, 495538.[ISI][Medline]
Jones,E.Y., Davis,S.J., Williams,A.F., Harlos,K. and Stuart,D.I. (1992) Nature, 360, 232239.[CrossRef][ISI][Medline]
Kawasaki,H. and Kretsinger,R.H. (1995) Protein Profile 2, 297490.[Medline]
Kilhoffer,M.C., Gerard,D. and Demaille,J.G. (1980) FEBS Lett., 120, 99103.[CrossRef][ISI][Medline]
Koch,A.W., Pokutta,S., Lustig,A. and Engel,J. (1997) Biochemistry, 36, 76977705.[CrossRef][ISI][Medline]
Kraulis,P.J. (1991) J. Appl. Crystallogr., 24, 946950.[CrossRef][ISI]
Linse,S. and Forsen,S. (1995) Adv. Second Messenger Phosphoprotein Res., 30, 89151.[ISI][Medline]
Linse,S., Brodin,P., Johansson,C., Thulin,E., Grundstrom,T. and Forsen,S. (1988) Nature, 335, 651652.[CrossRef][ISI][Medline]
Lu,Y. and Valentine,J.S. (1997) Curr. Opin. Struct. Biol., 7, 495500.[CrossRef][ISI][Medline]
Marsden,B.J., Shaw,G.S. and Sykes,B.D. (1990) Biochem. Cell. Biol., 68 (3), 587601.[ISI][Medline]
Pinto,A.L., Hellinga,H.W. and Caradonna,J.P. (1997) Proc. Natl Acad. Sci. USA, 94, 55625567.
Pokutta,S., Herrenknecht,K., Kemler,R. and Engel,J. (1994) Eur. J. Biochem. 223, 10191026.[Abstract]
Rajini,B., Shridas,P., Sundari,C.S., Muralidhar,D., Chandani,S., Thomas,F. and Sharma,Y. (2001) J. Biol. Chem., 276, 3846438471.
Regan,L. (1995) Trends Biochem. Sci., 20, 280285.[CrossRef][ISI][Medline]
Shea,M.A., Verhoeven,A.S. and Pedigo,S. (1996) Biochemistry, 35, 29432957.[CrossRef][ISI][Medline]
Tanford,C. (1968) Adv. Protein Chem., 23, 121282.[Medline]
Toma,S., Campagnoli,S., Margarit,I., Gianna,R., Grandi,G., Bolognesi,M., Filippis, I.D. and Fontana,A. (1991) Biochemistry, 30, 97106.[ISI][Medline]
Wang,C.L., Aquaron,R.R., Leavis,P.C. and Gergely,J. (1982) Eur. J. Biochem., 124, 712.[Abstract]
Wu,X. and Reid,R.E. (1997) Biochemistry, 36, 36083616.[CrossRef][ISI][Medline]
Yang,J.J., Caroll,A.R., Yang,W., Ye,Y.M. and Nguyen,C. (2000a) Cell Biochem. Biophys., 33, 253273.[ISI][Medline]
Yang,J.J., Yang,H.D., Ye,Y.M., Hopkins,H. and Hastings,H. (2000b) Cell Biochem. Biophys., 36, 118.
Yang,W., Lee,H.W., Pu,M., Hellinga,H. and Yang,J.J. (2000a) In Computational Studies, Nanotechnology and Solution Thermodynamics of Polymer Systems. Kluwer Academic/Plenum Publishers, New York.
Yang,W., Tsai,T., Kats,M. and Yang,J.J. (2000b) J. Pept. Res., 55, 203215.[CrossRef][ISI][Medline]
Yang,W., Lee,H.W., Hellinga,H. and Yang,J. (2002) Proteins, 47, 344356.[CrossRef][ISI][Medline]
Ye,Y.M., Lee,H.W., Yang,W., Shealy,S.J., Wilkins,A.L., Liu,Z.R. and Yang,J.J. (2001) Protein Eng., 14, 10011003.
Received September 28, 2001; revised February 26, 2002; accepted April 1, 2002.