pip-on-chip-label-free-study-protein-phosphoinositide

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

The Pennsylvania State University

The interactions of two highly positively charged short peptide sequences with negatively charged lipid bilayers were explored by fluorescence binding assays and all-atom molecular dynamics simulations. The bilayers consisted of mixtures of phosphatidylglycerol (PG) and phosphatidylcholine (PC) lipids as well as a fluorescence probe that was sensitive to the interfacial potential. The first peptide contained nine arginine repeats (Arg9), and the second one had nine lysine repeats (Lys9). The experimentally determined apparent dissociation constants and Hill cooperativity coefficients demonstrated that the Arg9 peptides exhibited weakly anticooperative binding behavior at the bilayer interface at lower PG concentrations, but this anticooperative effect vanished once the bilayers contained at least 20 mol % PG. By contrast, Lys9 peptides showed strongly anticooperative binding behavior at all PG concentrations, and the dissociation constants with Lys9 were approximately 2 orders of magnitude higher than with Arg9. Moreover, only arginine-rich peptides could bind to the phospholipid bilayers containing just PC lipids. These results along with the corresponding molecular dynamics simulations suggested two important distinctions between the behavior of Arg9 and Lys9 that led to these striking differences in binding and cooperativity. First, the interactions of the guanidinium moieties on the Arg side chains with the phospholipid head groups were stronger than for the amino group. This helped facilitate stronger Arg9 binding at all PG concentrations that were tested. However, at PG concentrations of 20 mol % or greater, the Arg9 peptides came into sufficiently close proximity with each other so that favorable like-charge pairing between the guanidinium moieties could just offset the long-range electrostatic repulsions. This led to Arg9 aggregation at the bilayer surface. By contrast, Lys9 molecules experienced electrostatic repulsion from each other at all PG concentrations. These insights may help explain the propensity for cell penetrating peptides containing arginine to more effectively cross cell membranes in comparison with lysine-rich peptides.

Polyarginine Interacts More Strongly and Cooperatively than Polylysine with Phospholipid Bilayers.

The orchestrated recognition of phosphoinositides and concomitant intracellular release of Ca(2+) is pivotal to almost every aspect of cellular processes, including membrane homeostasis, cell division and growth, vesicle trafficking, as well as secretion. Although Ca(2+) is known to directly impact phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca(2+) with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI(4,5)P2 containing liposomes reveal that Ca(2+) as well as Mg(2+) reduce the zeta potential of liposomes to nearly background levels of pure phosphatidylcholine membranes. Strikingly, lipid recognition by the default PI(4,5)P2 lipid sensor, phospholipase C delta 1 pleckstrin homology domain (PLC δ1-PH), is completely inhibited in the presence of Ca(2+), while Mg(2+) has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca(2+) binding to the PI(4,5)P2 headgroup and carbonyl regions leads to confined lipid headgroup tilting and conformational rearrangements. We rationalize these findings by the ability of calcium to block a highly specific interaction between PLC δ1-PH and PI(4,5)P2, encoded within the conformational properties of the lipid itself. Our studies demonstrate the possibility that switchable phosphoinositide conformational states can serve as lipid recognition and controlled cell signaling mechanisms.

Article	Year
Polyarginine Interacts More Strongly and Cooperatively than Polylysine with Phospholipid Bilayers. The journal of physical chemistry. B\| PubMed ID: 27571288	2016
Calcium Directly Regulates Phosphatidylinositol 4,5-Bisphosphate Headgroup Conformation and Recognition. Journal of the American Chemical Society\| PubMed ID: 28177616	2017

Simou Sun

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