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Anal. Chem. 1993, 65,2676-2678
Engineering Protein Orientation at Surfaces To Control Macromolecular Recognition Events Mark A. McLean, Patrick S . Stayton, and Stephen G. Sligar' Beckman Institute of Advanced Science and Technology, 405 North Mathews Street, Urbana, Illinois 61801
In this report we demonstrate that heterologous
modeling studies generated from the isolated X-ray crystal structures first noted the striking electrostatic complementarity of charged amino acids surrounding the exposed heme edges of both proteins.13 The basic tenants of the computer model have been largely confirmed through chemical modification and genetic engineering studies.14J5 The complementary binding interface places the two heme edges in close proximity for fast electron transfer between the redox INTRODUCT1ON centers.l6J7 Genetic engineering techniques have been utilized to Surface-immobilized proteins have had a great impact in independently place a unique thiol side chain at amino acid many fields of basic research and in many industrial and positions 65 and 8.l8 Position 65 was chosen to provide an medical technologies.'v2 Most research in this area has been attachment site close to the proposed cytochrome b5/ directed toward controlling the overall activity of the hybrid cytochrome c binding interface, yet spatially distinct enough biomaterials, for example, through reversibly affecting the surrounding matrix properties3 or by immobilizing related to prevent steric interference. In solution, labeling this site proteins in close proximity to provide multistep enzyme with the fluorophore acrylodan permits an independent processing.4 In principle, a great leap in biotechnological measurement of the equilibrium constant,l8 which closely potential would be realized if the immobilization process itself agrees with absorption difference titration^.'^ This finding, specified self-assembly and regulated function, but very little and a separate study utilizing the T65C mutant as a labeling research has been aimed at selectively controlling molecular site for a ruthenium redox center used to photoinitiate electron recognition events at the protein-substrate i n t e r f a ~ e . ~ , ~ transfer from cytochrome b5 to cytochrome c,17 strongly Because many molecular recognition processes are controlled support the hypothesis that labeling this position does not through specificity in complementary reactive surfaces, sterically inhibit complex formation. The TSC mutant controlling the orientation of immobilized proteins is perhaps positions the unique cysteine residue on the surface opposite the most straightforward means toward manipulating asthe exposed heme edge, at a site diametrically opposite the sembly and function in this manner. We have recently binding interface. These two mutants have been used to build demonstrated that proteins in monolayer assemblies can be monolayer protein assemblies on functionalized glass subdifferentially oriented on planar surfaces by genetically strates. Linear dichroism measurements of the prosthetic engineering unique attachment sites,7and here we show that heme group orientation demonstrate that T65C and TSC are protein-protein interactions can be controlled at an immodifferentially oriented at the substrate interface.' These bilization interface by similarly controlling the orientation of results suggested that designed attachment sites could be the protein binding surface. Protein-protein interactions are used to control the orientation of the cytochrome bs binding crucial t p important biological and biotechnological processes surface relative to a stationary support. Here we report the such as antibody-based diagnostics, affinity chromatography, control of macromolecular recognition between cytochrome electron transfer, and vectoral proton transfer. Manipulating b5 and cytochrome c through specific orientation on a macromolecular specificity at interfaces is thus an important chromatography support. step toward controlling function and self-assembly in a wide variety of immobilized protein systems. MATERIALS AND METHODS One of the best studied protein-protein complexes is the cytochrome bdcytochrome c electron-transfer pair. Extensive Site-Directed Mutagenesis. The cytochrome bg TSC and biochemical, biophysical, theoretical, and genetic investigaT65C mutations were generated as previously describedl8 and tions have provided a detailed understanding of the interaction surfaces for both cytochromes bb and c.b12 Computer
protein-protein interactions can be controlled at an immobilization surface by genetically engineering an appropriately de novo designed attachment point on the protein surface.
* Address correspondence to this author. (1)Methods in Enzymology; Mosbach, K., Ed.; Academic Press: New York, 1976; Vol. 44. (2) Langer, R. Science 1990, 249, 1527. (3) Hoffman, A. S. MRS Bull. 1991, 16, 42-46. (4) Mosbach, K.; Mattiasson, B. In Methods in Enzymology;Mosbach, K., Ed.; Academic Press: New York, 1976; Vol. 44, pp 453-478. (5) Whitesides, G. M.; Mathias, J. P.; Seto, C. T. Science 1991, 254, 1312-1319 (6) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (7) Stayton, P. S.; Olinger, J. M.; Bohn, P. W.; Sligar, S. G. J. Am. Chem. SOC.1992, 114,9298-9299, (8) Rodgers, K. K.; Pochapsky, T. C.; Sligar, S. G. Science 1988,240, 1657-1659. (9) Ferguson-Miller, S.; Brautigan, D. L.; Margoliash, E. J.B i d . Chem. 1978,253, 149-159.
(10) Dailey, H. A.; Strittmatter, P. J. Biol. Chem. 1979, 254, 53885396. (11)Wendoloski, J. J.; Matthew, J. B.; Weber, P. C.; Salemme, F. R. Science 1987, 238, 794-797. (12) Hazzard,J.T.;Mclendon,G.;Cusanovich,M.A.;Das,G.;Sherman, F.; Tollin, G. Biochemistry 1988, 27, 4445-4451. (13) Salemme, F. R. J . Mol. B i d . 1976,102, 563-568. (14) Kang, C. H.;Brautigan, D. L.; Osheroff, N.; Margoliash, E. J.Biol. Chem. 1978, 253, 6502-6510. (15) Rodgers, K. K.; Sligar, S. G. J . Mol. Biol. 1991,221, 1453-1460. (16) Mclendon, G.; Miller, J. R. J. Am. Chem. SOC.1985, 107, 78117816. (17) Willie, A.; Stayton, P. S.; Durham, B.; Sligar, S. G.; Millet, F. Biochemistry 1992, 31, 7237-7243. (18)Stayton,P. S.; Fisher, M. T.; Sligar, S. G. J . Biol.Chem. 1988,263, 13544-13548. (19) Mauk, M. R.; Reid, L. S.; Mauk, A. G. Biochemistry 1982, 21, 1843-1846.
0003-2700/93/0365-2676$04.00/00 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65. NO. 19. OCTOBER 1. 1993 2677
! l 61I5 F c
0 .-
I
c
m
I
a,
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I
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0
D66C Or
10
20
40
30
50
Conc cyt b5 pM Figure 2 Cvtochrome c column retention times as a function of Cytochrome &-T~cconcentration on the Immobilized support.
Figure 1. Schematic of cytochrome bs immoblllzation system,
purified as described by von Bodman et a1.'8 In addition to the T8C and T65C mutant proteins, adouble mutant with attachment sites on either side of the heme edge was constructed (D66C/ E380 using a protocol slightly modified from the method of Sligar et aLZ1 Briefly, two oligonucleotide primers encoding the position 66 mutation and the position 38 mutation were used to amplify the gene segment between the mutation sites. This product was then utilized as a primer with the universal sequencing primer to amplify the 3'-end of the mutated gene. Finally, this secondary product was used with the reverse sequencing primer to amplify the entire gene. This mutant, like T65C, places the attachment sites relativelyclose to, yet sterically distinct from, the binding interface. The double mutant has two free thiolsforimmobilization, although the percentageofnrotein linked at both sites is undetermined. Preparation of Cytochrome b5 Affinity Resin. Specific sulfhydryl mutations, TBC, T65C, D66C/E38C, of cytochrome bS were reducedfor lOminwitha1:1ratiodithiothreitolinl00mM NaPi, pH 7.8. The proteins were labeled by adding iodoacetylLC-biotin obtained from Pierce at a 21 ratio of biotin to protein. The reaction was allowed to proceed overnight. Excess biotin label was removed by passing the sample over a Sephadex G-25 column equilibrated in 100 mM NaPi, pH 7. Aliquots of the hiotinylated sample(s) were slowly added to a slurry of avidinSepharme 6B resin obtained from Pierce. After washing extensively with buffer, the concentrations of the cytochrome bs on the column were measured by taking the absorbance of a l(t 20% slurry in a Hitachi 3800 spectrophotometer equipped with an integrating sphere. The slurry was stirred and a spectrum was taken before an appreciable amount of settling could occur. The concentration was determined by using an extinction coefficient of 130mM-'at 412 nm." Since the active protein has the heme incorporated, we can take the 412-nm absorbance as a representation of the amount of active cytochrome bs hound to the solid support. A schematic representation of the cbromatographic supports that were constructed is shown in Figure 1. (20) Beck von Bodman, S.; Schuler. M. A,; Jollie, D. R.;Slip-, S. G . R o c . Natl. Acad. Sei. U S A . 1986,83,9443-9441. (21) Sligar, S. G.; Filipovic, D.; Stsyton, P. S. In Methods i n Enzymology; Waterman, M. R.,Johnson, E. F., Eds.; Academic Press: New York, 1992: Vol. 206, pp 31-49. (22) Margoliash, E.; Frowhirt, N. Biochem. J. 1959, 71,57*572.
Time (min) F W r e 3. OrleniaMn+pedficequilibrium binding IMJmrms. Equllbrlum binding isothermswere determined from the elution profiles generated
-
.
with linear ionic strength gradients: (- -)T8C. (- -) T65C, (.) E38G and (-) avidin control.
D66CI
Affinity Chromatography of Cytochrome e. Horse heart cytochrome e, Type VI, obtained from Sigma waa used without further purification. A 206 fiM solution of cytochrome c was prepared in 1 mM NaPi, pH 7.4. The affinity columns were packedintoaPharmaciaHR5/5columninwhichthe totalvolume was 0.8 mL. The column was connected to a Pharmacia FPLC pump system and equilibrated in 1mM Napi, pH 7.4, by passing seven column voiumes through at 0.5 mL/min. A 50-pL aliquot of the cytochrome c solution was loaded onto the column, and the column was washed with one column volume of buffer. A gradient was run from 1mM NaPi, pH 7.4, to 1mM NaPJlM) mM NaCI, pH 7.4, over a total volume of 30 mL with a flow rate of 0.5 mL/min. The elution of cytochrome c was monitored by the ahsorhanceat405nm. TheT8C cytochrome bsconcentration dependence was determined inorder tocorrect forthedifferences in concentration between the columns containing the mutant cytochrome bg. It is expected that there would be a linear relationship in the differences in retention times of cytochrome c vs cytochrome bs concentration, and the slope of this line will he proportional to the binding constant between cytochrome b6 and cytochrome c.23.u We can thus correct for the concentration differencesinthe columns. TheT8C mutation was used toderive the correction factor because its reactive surface is oriented away from the solid support and exhibits the longest retention time. The retention times of mutant binding isotherms are corrected to represent a concentration that is equal to that of the T8C column. The binding isotherms were generated by integrating the elution profile over time and normalizing them to the total area. This curve was then subtracted from 1to get the fraction of Cytochrome c retained on the column. (23) Hethcote, H. W.; Delisi, C. J. Chromatogr. 1982,248, 183-202. (24) Delisi, C., Hethcote,H. W. Affinity ChrDmotogrnphynndReloted Techniques;A d . Chem. Symp. Ser. 1982,9,63-78.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 19, OCTOBER 1, 1993
RESULTS The binding isotherms for the T65C, T8C, and D66C/E38C mutants are shown in Figure 3. The striking dependence of cytochrome c affinity on a cytochrome b5 attachment site is readily observed. Consistent with the previous measurement of differential T8C and T65C orientations at surface immobilization sites, these mutants display distinct binding isotherms. Further, the reduction in cytochrome c affinity with the T65C and D66C/E38C mutants is in the direction expected for orienting the cytochrome b5 binding surface toward the immobilization interface and inhibiting interaction with the process stream, while the T8C data are consistent with an orientation allowing for substantial cytochrome c interaction. Control measurements of the binding isotherm for cytochrome c-avidin interactions show that the T65C and D66CfE38C association constants are higher than any nonspecific background, indicating that a majority of the cytochrome bJcytochrome c interactions are retained. These results, in combination with the previous documentation indicating cytochrome c binds close to, but sterically distinct (25) Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; W. H. Freeman and Co.: New York, 1985; Chapter 10.
from, the T65C and D66C/E38C positions, strongly suggests that orientation-dependent molecular recognition has been achieved in this immobilized protein system. Orientational control of protein-protein molecular recognition at interfaces will be important in two crucial and interrelated aspects of process design: protein function and molecular assembly. Protein function is often exquisitely sensitive to the spatial relationships of subunits and partner pr0teins.~5This work demonstrates the ability to control molecular orientation of individual protein molecules, allowing for the possibility of constructing patterned multilayer protein assemblies where function is controlled by the molecular architecture specified in the availability of reactive surfaces.
ACKNOWLEDGMENT Supported by NIH Grants GM 31756 and GM 33775 and the Biotechnology Research and Development Corp.
RECEIVEDfor review June 1, 1993. Accepted June 25, 1993.
