Deletion of the helical motif in the intestinal fatty acid-binding protein reduces its interactions with membrane monolayers: Brewster angle microscopy, IR reflection-absorption spectroscopy, and surface pressure studies.
Intestinal fatty acid binding protein (IFABP) appears to interact directly with membranes during fatty acid transfer [Hsu, K. T., and Storch, J. (1996) J. Biol. Chem. 271, 13317-13323]. The largely alpha-helical "portal" domain of IFABP was critical for these protein--membrane interactions. In the present studies, the binding of IFABP and a helixless variant of IFABP (IFABP-HL) to acidic monolayers of 1,2-dimyristoylphosphatidic acid (DMPA) has been monitored by surface pressure measurements, Brewster angle microscopy (BAM), and infrared reflection-absorption spectroscopy (IRRAS). Protein adsorption to DMPA 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 of lipid-containing domains. IFABP exhibited more rapid changes in both phases than IFABP-HL. The second phase was absent when IFABP interacted with zwitterionic monolayers of 1,2-dipalmitoylphosphatidylcholine, revealing the important role of electrostatics at this stage. BAM images of DMPA monolayers with either protein revealed the formation of domains leading eventually to rigid films. Domains of DMPA/IFABP-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 substantially stronger ordering effect induced by IFABP. The physical measurements thus suggested differing degrees of direct interaction between the proteins and DMPA monolayers with the IFABP/DMPA interaction being somewhat stronger. These data provide a molecular structure rationale for previous kinetic 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].
Wu
F
F
Department of Chemistry, Newark College of Arts and Science, Rutgers University 73 Warren Street, Newark New Jersey 07102, USA.
Corsico
B
B
Flach
C R
CR
Cistola
D P
DP
Storch
J
J
Mendelsohn
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D020404:Glycerophospholipids / Q000737:chemistry; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D008563:Membrane Lipids / Q000737:chemistry*; D008853:Microscopy / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D017433:Protein Structure, Secondary / Q000235:genetics; D051381:Rats; D017384:Sequence Deletion; D013055:Spectrophotometry, Infrared / Q000379:methods; D013499:Surface Properties
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D020404:Glycerophospholipids; D008563:Membrane Lipids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C025602:dimyristoylphosphatidic acid