X-ray Scattering Studies of Maquette Peptide Monolayers. 2. Interferometry at the Vapor/Solid Interface Joseph Strzalka,* ,² Xiaoxi Chen, ‡ Christopher C. Moser, ‡ P. Leslie Dutton, ‡ John C. Bean, § and J. Kent Blasie ² Department of Chemistry and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Electrical Engineering, University of Virginia, Charlottesville, Virginia 22904 Received July 1, 2000. In Final Form: November 2, 2000 We apply X-ray interferometry to study the profile structure of Langmuir-Blodgett (LB) monolayers containing maquette peptides, de novo di-R-helical synthetic peptides designed as model systems for studying biological electron transfer. The results demonstrate that it is possible to create monolayers with the peptide vectorially oriented with its helical axis (the direction of electron transfer within the holopeptide) approximately normal to the surface of the solid support. This orientation can even be achieved when the orientation of the peptide in the precursor Langmuir film at the air/water interface is parallel to the surface, indicating that reorganization of the monolayer can occur during or after LB deposition. Though issues regarding the low density of the film and variability between samples remain to be addressed, the work represents an important step toward future correlated functional/structural studies of these peptides. Introduction Maquette peptides form the basis of an approach to understanding the behavior of redox protein complexes, which perform an array of vital functions including respiration and photosynthesis. Redox protein complexes are typically large and membrane-bound and, hence, difficult to purify and study. In contrast, synthetic maquette peptides are smaller, simpler, soluble peptides designed to mimic a part of the structure and function of natural redox complexes. We seek to understand proteins better by constructing our own. Inspired by part of the transmembrane domain of cytochrome bc 1 , the four-helix bundle motif comprises the basic structural design of one family of maquettes. 1 In the prototype, a 31-mer peptide is synthesized, designed as 27 residues forming an amphipathic R-helix and the other residues a short, flexible loop ending in a cysteine residue at the N-terminus of the peptide. In solution, a disulfide bond forms between the cysteines of two helices, forming a dihelical unit. The hydrophobic effect apposes the two helices, forming a pair of bis-His binding sites for prosthetic groups between the helices, and further drives the association of dihelical units into four-helix bundles. Structural and functional characterization of the pep- tides plays a central role in the maquette peptide program. It is important to compare the product synthesized with the intended design and to compare its functions with those of natural proteins. For the electron transfer properties of proteins, these two aspects of characterization are coupled, as the determination of a rate of electron transfer must be accompanied by knowledge of the distance and medium through which it occurs to be most meaningful. X-ray interferometry on oriented monolayers can provide the profile structure, the average structure of the monolayer projected onto the coordinate normal to its surface, and also preserve the peptide in an environ- ment in which its function can also be studied. For the most thorough characterization, the peptide should ideally be oriented so that the direction of electron-transfer coincides with the normal to the monolayer surface, the direction probed by interferometry. Part 1 of this study demonstrated that the orientation of maquette peptides in a monolayer at the air/water interface can be controlled via the macroscopic parameter π, the surface pressure of the film. 2 Although the order of Langmuir films is not always maintained upon transfer to a solid support, 3-5 the finding suggests that Langmuir-Blodgett (LB) depo- sition of maquette peptide films may produce a suitably ordered film. In this study, we spread monolayers of two different maquette peptides, the original prototype and a variant with a C 16 saturated hydrocarbon chain coupled to the free amine group of the N-terminal cysteine of each R-helix. Using Langmuir-Blodgett techniques, we transfer the monolayers from the air/water interface to solid supports incorporating an inorganic reference structure, and in- vestigate the profile structure of these films using X-ray interferometry. In an important step toward future correlated structural/functional studies, we demonstrate that in each system the peptide does orient within the monolayer with the long axis of the helices, presumably the direction of electron transfer between the prosthetic group binding sites, aligned approximately along the normal to the surface of the support. Differences between the expected and observed profile structures and implica- tions for future work are also discussed. * To whom correspondence may be addressed: Department of Chemistry, Box 141, University of Pennsylvania, Philadelphia, PA 19104-6323; e-mail, strzalka@jkb2.chem.upenn.edu. ² Department of Chemistry, University of Pennsylvania. ‡ Department of Biochemistry and Biophysics, University of Pennsylvania. § Department of Electrical Engineering, University of Virginia. (1) Robertson, D. E.; Farid, R. S.; Moser, C. C.; Urbauer, J. L.; Mulholland, S. E.; Pidikiti, R.; Lear, J. D.; Wand, A. J.; DeGrado, W. D.; Dutton, P. L. Nature 1994, 368, 425-431. (2) Strzalka, J.; Chen, X.; Moser, C. C.; Dutton, P. L.; Bean, J.; Blasie, J. K. Langmuir 2000, 16, 10404. (3) Hann, R. A. Molecular Structure and Monolayer Properties. In Langmuir-Blodgett FIlms; Robserts, G., Ed.; Plenum Press: New York, 1990. (4) Riegler, J. E.; LeGrange, J. D. Phys. Rev. Lett. 1988, 21, 2492- 2495. (5) LeGrange, J. D. Phys. Rev. Lett. 1991, 66, 37-40. 1193 Langmuir 2001, 17, 1193-1199 10.1021/la0009285 CCC: $20.00 © 2001 American Chemical Society Published on Web 01/26/2001