Binding of substance P agonists to lipid membranes and to the neurokinin-1 receptor.
Three new analogues of the neuropeptide substance P (SP) were synthesized. The C-terminal message segment was made more hydrophilic in (Arg9)SP or more hydrophobic in (Nle9)SP. In (AcPro2, Arg9)SP the charge at the N-terminal address segment was reduced, while that of the message segment was increased. The rationale underlying these substitutions was to correlate the physical-chemical properties of the SP-analogues, in particular their lipid-induced conformation and membrane-binding affinity, with receptor binding and functional activity. In solution, all three analogues exhibited random coil conformations as evidenced by circular dichroism spectroscopy. Addition of SDS micelles induced partially alpha-helical structures. The same structure was also produced by negatively charged lipid vesicles for (AcPro2, Arg9)SP and (Arg9)SP whereas both alpha-helix-like structures and beta-sheet structures were observed for SP and (Nle9)SP. The measurement of the Gibbs adsorption isotherms and monolayer expansion studies provided quantitative data on the surface area requirement and on the membrane penetration area of the SP analogues. The thermodynamic parameters for lipid binding were determined with monolayer expansion for measurements and high-sensitivity titration calorimetry. The apparent binding constants, Kapp, for membranes containing 100% POPG were of the order of 10(3)- 10(5) M(-1). The binding was due to electrostatic attraction of the cationic peptides to the negatively charged membrane surface. The intrinsic (hydrophobic) binding constants, obtained after correcting for electrostatic effects, were much smaller with Kp=10+/- 1 M(-1) for (Arg9)SP, 9 +/- 1 M(-1) for (AcPro2, Arg9)SP, and 39 +/- 3 M(-1) for (Nle9)SP. The measurement of the binding affinities to the NK-1 receptor and of the in vitro activities showed that all three peptides behaved as agonists. Their binding affinity to the neurokinin-1 receptor decreased with the size of the side chains at position 9 of the amino acid sequence but was independent of the cationic charge of the peptides. The fact that even the highly charged (Arg9)SP has agonistic activity provides evidence that the binding epitope at the receptor is in a rather hydrophilic environment. This finding is in agreement with the low hydrophobic binding constants and the weak penetration of the three peptides into negatively charged membranes. It argues against a membrane mediated receptor mechanism and suggests that the agonist approaches the receptor binding, site from the aqueous phase.
Seelig
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A
University of Basel, Switzerland.
Alt
T
T
Lotz
S
S
Hölzemann
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006224:Cricetinae; D004563:Electrochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism*; D008823:Micelles; D008969:Molecular Sequence Data; D011312:Pressure; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D018040:Receptors, Neurokinin-1 / Q000737:chemistry / Q000378:metabolism*; D013373:Substance P / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D008081:Liposomes; D008563:Membrane Lipids; D008823:Micelles; D018040:Receptors, Neurokinin-1; D013373:Substance P