X-ray Scattering Studies of Maquette Peptide Monolayers. 1. Reflectivity and Grazing Incidence Diffraction at the Air/Water Interface Joseph Strzalka,* ,† Xiaoxi Chen, ‡ Christopher C. Moser, ‡ P. Leslie Dutton, ‡ Benjamin M. Ocko, § and J. Kent Blasie † Department of Chemistry and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Physics, Brookhaven National Laboratory, Upton, New York 11973 Received February 24, 2000. In Final Form: July 21, 2000 We present isotherm and X-ray reflectivity (XR) measurements from Langmuir monolayers of a de novo synthetic di-R-helical peptide, consisting of two identical 31-residue, mostly R-helical peptide units joined by a disulfide bond at their amino-termini. Fitting the XR data to slab models shows that the dihelices lie in the plane of the interface at low pressures. The monolayers were insufficiently stable for study at high pressures, but Langmuir films based on a derivative of the peptide alkylated at its amino termini permitted investigations over a larger range of pressures. We observed an orientational transition, in which the R-helices begin by lying in the plane of the interface at low surface pressures and orient themselves approximately normal to the interface at high pressures. We draw the same conclusions from the XR data when we analyze it using box refinement, an iterative, model-independent method for recovering structure from XR data. Mixtures of these palmitoylated peptides with a fatty acid (palmitic acid) or a phospholipid (DLPE) behaved similarly. None of the systems produced peaks in the grazing incidence diffraction signal indicative of long-range ordering of the upright R-helices. Off-specular in-plane scattering measurements based on the difference signal between the peptide/DLPE mixture and pure DLPE suggest that the peptide achieves only liquidlike order within the plane. We discuss the implications and prospects for future work on designed peptide monolayers incorporating prosthetic groups that could be used to study electron transfer in proteins and provide a basis for biomolecular electronics applications. Introduction Nature uses a variety of proteins with special prosthetic groups to control processes such as electron transfer in photosynthesis and oxidative phosphorylation. The com- plexity of the proteins involved defies easy investigations of the relationship between their structure and function. Recently, smaller de novo synthetic peptides have been developed whose composition and properties can be readily explored and modified as a stepping stone toward un- derstanding naturally occurring proteins. 1 These so-called “maquettes” provide model systems for testing theories of electron transfer in proteins. A family of maquettes based upon a four-helix bundle motif has been designed and synthesized. The general synthetic strategy utilizes a 31- residue peptide whose primary sequence is designed to be mostly an amphipathic R-helix except for three glycine and one cysteine residues at the amino terminus. The side-chain sulfhydryl group of the N-terminal cysteines dimerizes via a disulfide linkage to form a dihelical unit which then self-assembles in isotropic aqueous solution to form a four-helix bundle. Histidine residues at apposed positions in each helix of the dihelical unit can then be utilized for bis-histidyl ligation of metalloporphyrin prosthetic groups at one or more sites in the primary sequence. Heme groups bound to these sites have been demonstrated to have spectral and electrochemical prop- erties closely resembling those of redox proteins found in nature. In order for meaningful data to be extracted from electrochemical measurements, experimenters need to know the structure of the molecule so that electron- transfer rates can be related to the distance and medium through which the transfer occurs. X-ray and neutron studies performed on thin-film samples can provide this key information. A thin, monomolecular film of protein molecules cover- ing a planar electrode comprises the simplest and most effective geometry for electron transfer studies and also constitutes an appropriate specimen for reflectivity mea- surements, making correlated structural/functional stud- ies possible. Once the electron-transfer properties of maquettes and the means to design desired properties are sufficiently understood, this geometry offers a basis for biomolecular electronics applications. We set as our goal the creation of dense, well-ordered films of maquette peptides on solid supports. We con- centrated on Langmuir-Blodgett techniques for achieving such a film since control of the surface pressure of the precursor Langmuir monolayer affords us a macroscopic means of varying such microscopic properties as the density and orientation of the molecules in the film. The anisotropic nature of the interface can confine suitably designed amphiphilic molecules to the plane of the interface and orient them vectorially. This may in turn may provide a suitable environment for further organiza- tion, such as two-dimensional crystallization or the directed incorporation of prosthetic groups to particular binding sites. Due to advances in synchrotron X-ray scattering techniques, we were able to study directly the * To whom correspondence may be addressed: Department of Chemistry, Box 141, University of Pennsylvania, Philadelphia, PA 19104-6323; strzalka@jkb2.chem.upenn.edu. † Department of Chemistry, University of Pennsylvania. ‡ Department of Biochemistry and Biophysics, University of Pennsylvania. § Department of Physics, Brookhaven National Laboratory. (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. 10404 Langmuir 2000, 16, 10404-10418 10.1021/la000264z CCC: $19.00 © 2000 American Chemical Society Published on Web 12/01/2000