Binding of acylated peptides and fatty acids to phospholipid vesicles: Pertinence to myristoylated proteins

Peitzsch, Robert M.; McLaughlin, Stuart

doi:10.1021/bi00090a020

Cited by 519 publications

(476 citation statements)

References 63 publications

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“…A protein standard curve is generated in parallel for each protein in order to determine what percentage of total added protein has pelleted with the vesicles. Molar partition coefficients (K), as defined by Peitzsch and McLaughlin [45] We have used this method extensively for studying binding of PH and FYVE domains to vesicles of different lipid compositions [20,46]. In these cases, the K D estimates agree very well with results obtained for binding to equivalent vesicles from ITC (for PLC 1 ) and surface plasmon resonance studies (see Section 2.3.3.5).…”

Section: Vesicle-binding Assaymentioning

confidence: 53%

Determining selectivity of phosphoinositide-binding domains

Narayan

Lemmon

2006

Methods

119

121

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The burgeoning of phosphoinositide-binding domains and proteins in cellular signaling and trafficking has drawn laboratories from a wide variety of fields into the study of lipid interactions with peripheral membrane proteins. Many different approaches have been developed to assess phosphoinositide binding, some of which are more problematic than others, and some of which can be quantitated more readily than others. With a focus on the methods used in our laboratory, we describe here the considerations that need to be taken into account when establishing -and quantitating -the specific binding of a protein or domain to phosphoinositides in membranes. We also discuss briefly a few examples in which no clear consensus has yet been reached as to the specificity of a given domain or protein because of discrepancies between different commonlyused approaches.

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Section: Vesicle-binding Assaymentioning

confidence: 53%

Determining selectivity of phosphoinositide-binding domains

Narayan

Lemmon

2006

Methods

119

121

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“…We use a molar partition coefficient K (49,50) to describe the binding of the peptide to lipid vesicles without making assumptions about the absorption mechanism. The molar partition coefficient K is defined by the equation …”

Section: Materials-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholinementioning

confidence: 99%

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

Wang

Gambhir

Hangyás-Mihályné

et al. 2002

Journal of Biological Chemistry

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204

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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.Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 plays many important roles in cells (reviewed in Refs. 1-8). Not only is PIP 2 the source of 3 second messengers, its hydrolysis via phospholipase Cs (PLCs) (9, 10) produces inositol 1,4,5-trisphosphate and diacylglycerol (11,12), and its phosphorylation via phosphoinositide 3-kinases produces phosphatidylinositol 3,4,5-trisphosphate (6, 13-15), but it also can be a second messenger itself (4,8,16,17). Moreover, PIP 2 helps regulate cytoskeletal attachment (18 -20), exo-and endocytosis (1-3), enzyme activity (21), and ion channel function (17,22). Several groups (2,8,16,23) have suggested that these myriad functions can be explained if there are different pools of PIP 2 in the plasma membrane. One hypothesis is that proteins act as reversible buffers to bind much of the PIP 2 and then release it locally in response to specific signals (8,24,25). These proteins would have to be present at a concentration comparable with PIP 2 , be localized to the plasma membrane, bind PIP 2 with high affinity, and release it in response to physiological stimuli. Myristoylated alanine-rich C kinase substrate (MARCKS) satisfies these criteria.MARCKS (reviewed in Refs. 26 -30), a ubiquitous protein kinase C (PKC) (31) substrate, is present at high concentration in many cell types (e.g. ϳ10 M in brain tissue (27, 32)) and is localized to the plasma membrane in quiescent cells. The binding of MARCKS to plasma membranes requires both hydrophobic insertion of its N-terminal myristate into the bilayer and electrostatic interactions between its effecto...

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“…In a series of biophysical investigations of the thermodynamics of insertion of lipid-modified model peptides in vesicles, functioning as model membranes, the contribution of the lipids to lipopeptide membrane affinity was determined. [45][46][47] This showed that an Nmyristoyl or an S-farnesyl group alone cannot contribute enough hydrophobic character to maintain stable membrane insertion of peptides, and therefore also of proteins.…”

Section: Methodsmentioning

confidence: 99%