Biomedical subjects

Alok Gambhir

Publications and source records attributed to Alok Gambhir.

6 recordsLinked to original sources

PubMed2006

Membrane-bound basic peptides sequester multivalent (PIP2), but not monovalent (PS), acidic lipids.

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.

Amino Acid Sequence↗

PubMed2004

A computational model for the electrostatic sequestration of PI(4,5)P2 by membrane-adsorbed basic peptides.

The multivalent acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) plays a key role in many biological processes. Recent studies show that unstructured clusters of basic residues from a number of peripheral proteins can laterally sequester PI(4,5)P2 in membranes. Specifically, experiments suggest that the basic effector domain of the myristoylated alanine-rich C kinase substrate (MARCKS), or a peptide corresponding to this domain, MARCKS(151-175), sequesters several PI(4,5)P2 and that this sequestration is due to nonspecific electrostatic interactions. Here, we use the finite difference Poisson-Boltzmann method to test this hypothesis by calculating the electrostatic free energy of lateral sequestration of PI(4,5)P2 by membrane-adsorbed basic peptides: Lys-7, Lys-13, and FA-MARCKS(151-175), a peptide based on MARCKS(151-175). In agreement with experiments, we find that the electrostatic free energy becomes more favorable when: 1), Lys-13 and FA-MARCKS(151-175) sequester several PI(4,5)P2; 2), the linear charge density of the basic peptide increases; 3), the mol percent monovalent acidic lipid in the membrane decreases; and 4), the ionic strength of the solution decreases. In addition, the electrostatic sequestration free energy is in excess of the entropic penalty associated with localizing PI(4,5)P2. Our calculations, thus, provide a structural and quantitative description of the observed interaction of PI(4,5)P2 with membrane-adsorbed basic sequences.

Algorithms↗

PubMed2004

Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins.

The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of MARCKS reversibly sequesters a significant fraction of the L-alpha-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) on the plasma membrane. To test this, we utilize three techniques that measure the ability of a peptide corresponding to its effector domain, MARCKS(151-175), to sequester PIP2 in model membranes containing physiologically relevant fractions (15-30%) of the monovalent acidic lipid phosphatidylserine. First, we measure fluorescence resonance energy transfer from Bodipy-TMR-PIP2 to Texas Red MARCKS(151-175) adsorbed to large unilamellar vesicles. Second, we detect quenching of Bodipy-TMR-PIP2 in large unilamellar vesicles when unlabeled MARCKS(151-175) binds to vesicles. Third, we identify line broadening in the electron paramagnetic resonance spectra of spin-labeled PIP2 as unlabeled MARCKS(151-175) adsorbs to vesicles. Theoretical calculations (applying the Poisson-Boltzmann relation to atomic models of the peptide and bilayer) and experimental results (fluorescence resonance energy transfer and quenching at different salt concentrations) suggest that nonspecific electrostatic interactions produce this sequestration. Finally, we show that the PLC-delta1-catalyzed hydrolysis of PIP2, but not binding of its PH domain to PIP2, decreases markedly as MARCKS(151-175) sequesters most of the PIP2.

Amino Acid Sequence↗

PubMed2004

Fluorescence correlation spectroscopy studies of Peptide and protein binding to phospholipid vesicles.

We used fluorescence correlation spectroscopy (FCS) to analyze the binding of fluorescently labeled peptides to lipid vesicles and compared the deduced binding constants to those obtained using other techniques. We used a well-characterized peptide corresponding to the basic effector domain of myristoylated alanine-rich C kinase substrate, MARCKS(151-175), that was fluorescently labeled with Alexa488, and measured its binding to large unilamellar vesicles (diameter approximately 100 nm) composed of phosphatidylcholine and phosphatidylserine or phosphatidylinositol 4,5-bisphosphate. Because the large unilamellar vesicles are significantly larger than the peptide, the correlation times for the free and bound peptide could be distinguished using single color autocorrelation measurements. The molar partition coefficients calculated from the FCS measurements were comparable to those obtained from binding measurements of radioactively labeled MARCKS(151-175) using a centrifugation technique. Moreover, FCS can measure binding of peptides present at very low concentrations (1-10 nmolar), which is difficult or impossible with most other techniques. Our data indicate FCS can be an accurate and valuable tool for studying the interaction of peptides and proteins with lipid membranes.

Binding Sites↗

PubMed2002

Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions.

A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP(2)-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP(2). Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP(2) laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP(2) in the plasma membrane.

Amino Acid Sequence↗

PubMed2001

PIP(2) and proteins: interactions, organization, and information flow.

We review the physical properties of phosphatidylinositol 4,5-bisphosphate (PIP2) that determine both its specific interactions with protein domains of known structure and its nonspecific electrostatic sequestration by unstructured domains. Several investigators have postulated the existence of distinct pools of PIP2 within the cell to account for the myriad functions of this lipid. Recent experimental work indicates certain regions of the plasma membrane-membrane ruffles and nascent phagosomes-do indeed concentrate PIP2. We consider two mechanisms that could account for this phenomenon: local synthesis and electrostatic sequestration. We conclude by considering the hypothesis that proteins such as MARCKS bind a significant fraction of the PIP2 in a cell, helping to sequester it in lateral membrane domains, then release this lipid in response to local signals such as an increased concentration of Ca(++)/calmodulin or activation of protein kinase C.

Animals↗