Direct Enthalpy Measurements of the Calcium-Dependent Interaction of Annexin V and VI with Phospholipid Vesicles

Plager, Douglas A.; Nelsestuen, Gary L.

doi:10.1021/bi00249a010

Cited by 27 publications

(15 citation statements)

References 53 publications

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“…Initial experiments performed at high ratios of protein to membrane were consistent with earlier studies of annexin V from other groups (11,13). As shown in Fig.…”

Section: Comparison Of Enthalpy and Free Energy Values As A Function supporting

confidence: 88%

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Entropic and Enthalpic Contributions to Annexin V-Membrane Binding

Jeppesen

Smith

Gibson

et al. 2008

Journal of Biological Chemistry

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Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quantitatively elucidated. Analysis by isothermal microcalorimetry and calcium titration under conditions of low membrane occupancy showed that there was a strongly positive entropy change upon binding. For vesicles containing 25% phosphatidylserine at 0.15 M ionic strength, the free energy of binding was ؊53 kcal/mol protein, whereas the enthalpy of binding was ؊38 kcal/mol. Addition of 4 M urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quantitatively consistent with predictions from the WimleyWhite scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more positively charged, nor decreased by making it more negatively charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Analysis by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region makes an important contribution to overall binding affinity, supplementing the role of protein-calcium-phosphate chelates.

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“…Initial experiments performed at high ratios of protein to membrane were consistent with earlier studies of annexin V from other groups (11,13). As shown in Fig.…”

Section: Comparison Of Enthalpy and Free Energy Values As A Function supporting

confidence: 88%

“…Annexins bind to membranes with remarkably high affinity (8 -10), and the reaction is strongly exothermic (11)(12)(13), but the mechanistic basis for this reaction is complex. There is broad agreement on the importance of calcium for the interaction of annexins with membranes at neutral pH.…”

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confidence: 99%

Entropic and Enthalpic Contributions to Annexin V-Membrane Binding

Jeppesen

Smith

Gibson

et al. 2008

Journal of Biological Chemistry

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“…The presence of PE in the membrane has a large synergistic impact on function of PS for membrane binding by vitamin K-dependent proteins (24). Similar effects were seen for diverse proteins such as protein kinase C, annexins (25), pentraxins (26), myristoylated alanine-rich C kinase substrate (27), and blood clotting factor VIII (28).…”

Section: Factor VII (5)mentioning

confidence: 72%

Enhancement of Human Protein C Function by Site-directed Mutagenesis of the γ-Carboxyglutamic Acid Domain

Shen¹,

Shah²,

Dahlbäck³

et al. 1998

Journal of Biological Chemistry

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This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine. This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membrane contact but without further increase in membrane affinity at saturating calcium. Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant proteins and may contribute somewhat to higher function in these mutants. This family of mutants helped discriminate events that are necessary for protein-membrane binding. These include calcium binding to the free protein and subsequent protein-membrane contact. Depending on conditions of the assay used, the mutants displayed increased activity of the corresponding activated protein C (APC) derivatives. The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (؎ phosphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the largest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant activity of all proteins was stimulated by protein S, with the greatest impact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants were more active than normal APC. Protein C variants with enhanced function present new reagents for study of coagulation and may offer improved materials for biomedical applications.

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“…Many parameters may influence the surface concentration of annexin, e.g. the calcium concentration (43,44), transmembrane potential (45), membrane composition (46) etc.…”

Section: Discussionmentioning

confidence: 99%