Deletion of the Helical Motif in the Intestinal Fatty Acid-Binding Protein Reduces Its Interactions with Membrane Monolayes: Brewster Angle Microscopy, IR Reflection-Absorption Spectroscopy, and Surface Pressure Studies

摘要

Intestinal fatty acid binding protein (IF ABP) appears to interact directly with membranes during fatty acid transfer [Hsu, K. T., and Storch, J. (1996) J. BioI. Chem. 271, 13317-13323]. The largely [X.-helical "portal" domain of IFAHP was critical for these protein-membrane interactions. In the present studies, the binding of IFAHP and a helixless variant of IFABP (IFAHP-HL) to acidic monolayers of 1,2-dimyristoylphosphatidic acid (DMPA) has been monitored by surface pressure measurements, Hrewster angle microscopy (HAM), and infrared reflection-absorption spectroscopy (IRRAS). Protein adsorption to DMP A exhibited a two phase kinetic process consisting of an initial slow phase, arising from protein binding to the monolayer and/or direct interfacial adsorption, and a more rapid phase that parallels formation Jf lipid-containingdomains. IFAHP exhibited more rapid changes in both phases than IFAHP-HL. The second phase was absent when IFAHP interacted with zwitterionic monolayers of 1,2-dipalmitoylphos- phatidylcholine, revealing the important role of electrostatics at this stage. HAM images of DMPA monolayers with either protein revealed the formation of domains leading eventually to rigid films. Domains Jf DMPA/IFAHP-HL formed more slowly and were less rigid than with the wild-type protein. Overall, the IRRAS studies revealed a protein-induced conformational ordering of the lipid acyl chains with a )ubstantially stronger ordering effect induced by IF A HP .The physical measurements thus suggested :liffering degrees of direct interaction between the proteins and DMPA monolayers with the IFABP/ DMP A interaction being somewhat stronger. These data provide a molecular structur. rationale for previous I<:inetic measurements indicating that the helical domain is essential for a collision-based mechanism of Fatty acid transfer to phospholipid membranes [Corsico, B., Cistola, D. P., Frieden, C. and Storch, J. '1998) Proc. Natl. Acad. Sci. U.S.A. 95. 12174-12178].