Papers

AVS1996 Session BI-MoA: Protein-Surface Interactions

Monday, October 14, 1996 1:30 PM in Room 203A

Monday Afternoon

Start	Invited?	Item
1:30 PM		BI-MoA-1 Biomolecular Architectures at Solid Substrates W. Knoll (Max-Planck-Institut f\um u\r Polymerforshung, Germany); M. Matsuzawa (Frontier Research Program, RIKEN, Japan); J. Ruehe (Max-Planck-Institut f\um u\r Polymerforschung, Germany) This contribution deals with the controlled build-up of supra-biomolecular architectures at functionalized surfaces. First, we report on some investigations of the binding of the protein streptavidin from solution to biotin as the recognition site linked to a membrane-mimetic surface on a solid support. Using surface plasmon spectroscopy as a highly sensitive optical technique we first concentrate on the functionalization of Au-surfaces by self-assembly monolayers (SAM) composed of thiols, sulfides and disulfides. We focus on the question of how to optimize the interface for maximum, molecularly controlled protein binding. Different strategies were developed by varying, in particular, the binding site density (through coadsorption of OH-functionalized molecules that dilute the lateral biotin density), the spacer length and the degree of disorder in the self-assembly monolayers. These concepts were then extended: Other protein systems were studied, e.g., the orientation-dependent binding between two cytochromes (c and b5), or various antibody-antigen interactions. Moreover, other biosystems like oligonucleotide hybidization reactions or the functional coupling of whole cells, e.g., hippocampal neuron cells to solid supports, were investigated. A particular biocompatibilization at solid surfaces play supported artificial membrane architectures. Various strategies based on either sequential adsorption and binding of the required components or on the spontaneous self- organization of specifically designed copolymers were developed.
2:10 PM		BI-MoA-3 Peptide-Antibody Recognition on Well Defined Surfaces Studied by Surface Plasmon Resonance V. Perez-Luna, M. O'Brien, K. Opperman, L. Tender (University of New Mexico); R. Harris (Commonwealth Biotechnologies, Inc.); G. Lopez (University of New Mexico) The kinetics and equilibrium conditions of antibody binding to surface immobilized peptides were studied by surface plasmon resonance (SPR). A rabbit polyclonal antibody against a peptide encompassing an epitope found at the N-terminal end of human \beta\-endorphin was prepared. Then a family of peptides with varying affinities for the antibody were synthesized by replacing some residues within this epitope. These peptide sequences were extended at the N-terminus by covalent incorporation of biotynilated spacer arms. The spacer consisted of biotinyl-diaminodioxaoctanyl-succinyl-, which allowed the binding of peptides to surface immobilized streptavidin. Surface immobilization of streptavidin was accomplished by specifically binding streptavidin onto a biotin functionalized self-assembled monolayer (SAM). The SAM consisted of a biotin-terminated thiol, 11-mercaptoundecanoic-(8-biotinylamido-3,6-dioxaoctyl) amide, assembled on gold. Binding of streptavidin to this surface proceeded only through specific binding. When the biotin binding sites of streptavidin were blocked with a 100-fold excess of biotin, binding was not detected by SPR. Thus, only specific binding occurred on this SAM. The affinity of the peptides for the antibody in solution spanned a wide range (ca. 3 orders of magnitude). Thus allowing the detection of antibody binding in a wide concentration range. Antibody-peptide binding constants (Kd's) for the surface immobilized peptides where compared to Kd's determined in bulk solutions by isothermal titration calorimetry (ITC).
2:30 PM		BI-MoA-4 Molecular Recognition at the Protein-Biomineral Interface R. Clark (University of Washington); A. Campbell (Pacific Northwest National Laboratory); P. Stayton (University of Washington) Biological organisms exhibit sophisticated crystal engineering capabilities that underlie the remarkable material properties of mineralized tissues such as bone and nacre. While nature's biomineralization processes are a complex blend of finely controlled nucleation and growth events that are not currently well understood, it is known that organisms produce acidic proteins which play a key directoral role in controlling biological crystal growth. We have taken a systematic approach with a model protein to the question of how small, acidic proteins interact with biological crystals and control their growth rates. In particular, we were interested in defining the role of protein electrostatic surface properties in controlling secondary crystal growth. We report the first site-directed mutagenesis investigation into the relationship between protein surface charge distribution and crystal nucleation/growth. Our study demonstrates that the electrostatic surface charge distribution can directly determine whether biomineralization is promoted or inhibited. When preadsorbed to calcium oxalate monohydrate seed crystals at low surface coverages of 7%, the native protein with ten surface carboxylates significantly increases the rate of secondary precipitation and crystal growth compared to uncoated controls. A site-directed mutant with one large lobe of negative electrostatic surface charge removed (six carboxylates removed) displayed a complete reversal of this effect, as this engineered protein significantly inhibited the growth rate.
2:50 PM		BI-MoA-5 Biological Characterisation of Materials H. Nygren, C. Eriksson (University of G\um o\teborg, Sweden) Nonself recognition of foreign materials leads to well-known complications when exposing the human body to medical devices. The recognition process is an array of reactions ranging in time from milliseconds through years. The reaction array comprises several parts of general interest for the understanding of inflammatory processes at biomaterial surfaces. Of special interest is the interaction between platelets and plasma proteins, followed by platelet-neutrophil interaction and its later effect on macrophages. The adsorption of main plasma proteins (albumin, IgG, fibrinogen), activation of cascade enzymes (serine proteases) and adhesion of cells is measured with immunofluorescence techniques, using specific antibodies the binding of which is quantitated by computer-aided image analysis. The activation of cascade enzymes is detected with antibodies against thrombin (terminal step in the coagulation cascade), terminal complement component (TCC) and plasmin (terminal step in fibrinolysis). It is important to analyse terminal steps of the cascades, to ensure detection of activation. The activation of cells is measured by expression of selectin (platelet CD62), expresion of integrins (neutrophil CD11b), respiratory burst (NBT test) or formation of F-actin (phalloidin). Results will be presented from tests on TiO2, TiN, Gold and Graphite.
3:10 PM		BI-MoA-6 Immobilization and Patterning of Functional Proteins on Surfaces R. Brizzolara, J. Boyd, A. Tate (Naval Surface Warfare Center) The immobilization and patterning of functional proteins on surfaces impacts many applications such as biosensors and microelectromechanical systems. Often, these applications require the immobilization of the protein on a substrate with micron spatial resolution, in layers of controlled thickness, with a controlled orientation. Existing immobilization methods satisfy some of these requirements. The goal of the present work is the development of immobilization methods that satisfy all of the above requirements. In this paper we describe two such immobilization methods. We have used the light-transducing protein bacteriorhodopsin as a model protein in our investigation. In the first method, the immobilization is induced by genetic modification of bacteriorhodopsin, to facilitate a covalent attachment between protein and surface. XPS and AFM results on new variants, possessing multiple genetic modifications will be presented. In the second method, an antibody-directed technique will be described. AFM results demonstrating the use of this technique to immobilize bacteriorhodopsin through a specific antibody-antigen interaction will be presented, as well as results of work involving the use of this technique to pattern bacteriorhodopsin. We will compare these methods with other immobilization methods with regard to the above criteria. Extension of this work to other functional proteins will also be addressed. The Office of Naval Research and the NSWC Independent Research and Seed and Venture Programs are acknowledged for funding support. Many thanks to Professor Richard Needleman who assisted with the mutagenesis.
3:30 PM		BI-MoA-7 Functionalized Self-assembled Films to Control Biological Responses K. McClary, G. Mao, D. Grainger (Colorado State University) Because of the possibility to control surface chemistry, self-assembled organic films offer attractive opportunities to monitor interfacial responses in biological systems. Molecular determinants for protein adsorption and cell attachment to surfaces may be elucidated by surface chemistry studies presenting well-defined yet chemically variable interfaces. We have used derivatized alkylthiol monolayers as model materials interfaces in biological milieu. Results for two different systems will be presented. In the first, we used amino acid substituted mutants of the enzyme, T4 lysozyme, to study mutual influences of protein surface chemistry with monolayer surface chemistry. Each surface in this binding pair has an ability to change the adsorption kinetics and equilibrium adsorbed amounts as detected by in-situ ellipsometry. In the second system, fibroblast cells are grown on alkylthiol monolayers bearing distinct surface groups. Cell attachment, growth and expression of genetic information associated with growth responses are all shown to be influenced by surface chemistry. We attempt to correlate these responses to cell receptor binding and cell adhesion proteins that, in turn, are influenced by surface chemistry.
4:10 PM		BI-MoA-9 Correlation of Culture Stratum Composition and Cell Properties of Three Cell Types Grown on SAMS by XPS D. Jung, K. Bateman, M. Coulombe, R. Sathanoori, J. Hickman (Science Applications International Corporation); A. Schaffner, J. Barker (National Institutes of Health) We have previously shown that artificial surfaces comprised of silane self-assembled monolayers (SAMs) exhibit reproducible surface properties at the systems level and can be selected to maximize cell adhesion, survival and differention. We have found that the amount of protein deposited onto these artificial surfaces during culture can be correlated with morphological and electrical properties of the neurons. We use X-ray photoelectron spectroscopy (XPS) and null ellipsometry (NE) to measure and compare the amount of protein deposition by three cell types (primary embryonic hippocampal or spinal cord rat cells and a hybridoma neural cell line, NG108-15) cultured on SAM-modified surfaces in serum-free media. We have investigated the correlations of protein deposition with several culture conditions, media additives, and procedures with the objective of developing neuronal systems on artificial surfaces that show improved adhesion, survival, morphology, electrical excitability compared to conventional cultures using poorly defined biological surfaces (such as poly-D-lysine). Rat hippocampal and spinal cord neurons were cultured under a matrix of conditions including mechanical versus enzymatic (papain) dissociation, culture in serum followed by serum-free media, and the addition of growth factors. NG108-15 cell cultures were compared under conditions of frequent versus infrequent feeding, and with and without the addition of Hepes, which extends the buffer capacity of the medium.
4:30 PM		BI-MoA-10 Cysteine and Gly-Gly Adsorption and Desorption on Pt(111) P. L\um o\fgren, L. Hedberg (Chalmers University of Technology and G\um o\teborg University, Sweden); J. Lausmaa (Chalmers University of Technology and G\um o\teborg University); B. Kasemo (Chalmers University of Technology and G\um o\teborg University, Sweden) As model experiments for the interaction between biological molecules and solid surfaces we have started a series of adsorption/desorption studies of amino acids and simple peptides on Pt(111). The biological molecules are in situ deposited in UHV and characterized with TDS and XPS. Previously we have reported results on glycine and alanine. Here we report results from studies of the amino acid cysteine and the simplest dipeptide gly-gly. TDS results suggest that cysteine is deprotonated, presumably at the thiol group, upon adsorption . Around 300 K and above, the cysteine molecules in the first monolayer decompose and desorb in a complex series of reactions. With increasing coverages two cysteine desorption peaks are detected at 360 and 325 K, respectively. They are tentatively attributed to desorption from the second layer and from multilayers, respectively. The evaporation of gly-gly is sensitive to the sublimation temperature. Deposition of intact gly-gly molecules could be achieved at evaporation source temperatures below ~390 K while at higher temperatures significant amounts of decomposition products are detected in the adsorbed layers. The peptide molecules bound to the surface start to decompose around 350 K. No desorption of intact peptide molecules from the first monolayer can be detected. Peptide multilayer desorption is observed around 325 K. Results from a just initiated XPS study of these molecules will also be reported