Analysis of Myoglobin Adsorption to Cu(II)-IDA and Ni(II)-IDA Functionalized Langmuir Monolayers by Grazing Incidence Neutron and X-ray Techniques M. S. Kent,* H. Yim, and D. Y. Sasaki Department 1851, Sandia National Laboratories, Albuquerque, New Mexico 87185 S. Satija NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 J. Majewski Los Alamos Neutron Science Center, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 T. Gog Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439 Received November 24, 2003 The adsorption of myoglobin to Langmuir monolayers of a metal-chelating lipid in crystalline phase was studied using neutron and X-ray reflectivity (NR and XR) and grazing incidence X-ray diffraction (GIXD). In this system, adsorption is due to the interaction between chelated divalent copper or nickel ions and the histidine moieties at the outer surface of the protein. The binding interaction of histidine with the Ni-IDA complex is known to be much weaker than that with Cu-IDA. Adsorption was examined under conditions of constant surface area with an initial pressure of 40 mN/m. After ∼12 h little further change in reflectivity was detected, although the surface pressure continued to slowly increase. For chelated Cu 2+ ions, the adsorbed layer structure in the final state was examined for bulk myoglobin concentrations of 0.10 and 10 µM. For the case of 10 µM, the final layer thickness was ∼43 Å. This corresponds well to the two thicker dimensions of myoglobin in the native state (44 Å × 44 Å × 25 Å) and so is consistent with an end-on orientation for this disk-shaped protein at high packing density. However, the final average volume fraction of amino acid segments in the layer was 0.55, which is substantially greater than the value of 0.44 calculated for a completed monolayer from the crystal structure. This suggests an alternative interpretation based on denaturation. GIXD was used to follow the effect of protein binding on the crystalline packing of the lipids and to check for crystallinity within the layer of adsorbed myoglobin. Despite the strong adsorption of myoglobin, very little change was observed in the structure of the DSIDA film. There was no direct evidence in the XR or GIXD for peptide insertion into the lipid tail region. Also, no evidence for in-plane crystallinity within the adsorbed layer of myoglobin was observed. For 0.1 µM bulk myoglobin concentration, the average segment volume fraction was only 0.13 and the layer thickness was e25 Å. Adsorption of myoglobin to DSIDA-loaded with Ni 2+ was examined at bulk concentrations of 10 and 50 µM. At 10 µM myoglobin, the adsorbed amount was comparable to that obtained for adsorption to Cu 2+ - loaded DSIDA monolayers at 0.1 µM. But interestingly, the adsorbed layer thickness was 38 Å, substantially greater than that obtained at low coverage with Cu-IDA. This indicates that either there are different preferred orientations for isolated myoglobin molecules adsorbed to Cu-IDA and Ni-IDA monolayer films or else myoglobin denatures to a different extent in the two cases. Either interpretation can be explained by the very different binding energies for individual interactions in the two cases. At 50 µM myoglobin, the thickness and segement volume fraction in the adsorbed layer for Ni-IDA were comparable to the values obtained with Cu-IDA at 10 µM myoglobin. I. Introduction The interaction of biomacromolecules with lipid mem- branes is of great interest in many areas, including drug discovery, understanding cellular signaling, combating biowarfare agents, and the development of sensor materi- als. From a fundamental point of view, a general goal is to understand how various types of fundamental interac- tions, and their spatial distribution, affect the conforma- tion, orientation, and extent of aggregation (in-plane and out-of-plane of the lipid membrane) of an adsorbed protein. The orientation of an enzyme, for example, can affect access to its active site, and thus activity. Another important question is what conditions lead to denaturation upon adsorption to the membrane surface. In bulk solution, myoglobin has been shown to unfold at ambient pressure at 74 °C at pH 7.6, and at much lower temperatures at elevated pressures. 1 Myoglobin has also been found to unfold at pH 3.9 at ambient temperature and pressure 2,3 and even to form fibrils in the presence of sodium borate at pH 9.0 at 65 °C. 4 It is important to know whether significant conformational changes may occur upon strong adsorption to a substrate as well. The extent of penetration of protein segments into lipid membranes is a further topic of interest, particularly important for understanding the mechanisms by which toxins enter cells. Finally, it is important to understand the factors that control the ability of an ensemble of adsorbed proteins to sample the (1) Smeller, L.; Rubens, P.; Heremans, K. Biochemistry 1999, 38, 3816. (2) Bergers, J. J.; Vingerhoeds, M. H.; van Bloois, L.; Herron, J. N.; Janssen, L. H. M.; Fischer, M. J. E.; Crommelin, D. J. A. Biochemistry 1993, 32, 4641. (3) Puett, D. J. Biol. Chem. 1973, 248, 4623. 2819 Langmuir 2004, 20, 2819-2829 10.1021/la036207y CCC: $27.50 © 2004 American Chemical Society Published on Web 02/07/2004