Comparative Study of Analytical Techniques for Determining Protein Charge

Filoti, Dana I.; Shire, Steven J.; Yadav, Sandeep; Laue, Thomas M.

doi:10.1002/jps.24454

Cited by 46 publications

(95 citation statements)

References 27 publications

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“…Likewise, NMR spectroscopy revealed that neither K þ nor Na þ significantly perturbed PC liposome spectra (42), and estimates of the binding constants for Na , and Na þ ions. However, in general, ELS charge measurements for macromolecules the size of Nanodiscs have poor precision (15), which is evident in the ELS mobility data for PC vesicles, where the uncertainty nearly equals or exceeds the reported mobility (25).…”

Section: Polyelectrolyte Behavior Of Lipid Nanodiscsmentioning

confidence: 99%

“…The uncertainty in Z DHH from MCE measurements is estimated to be 56% (9,40), or 50.8 charge units for POPC Nanodiscs. Although it is difficult to estimate the uncertainty in the charge calculated from the amino acid composition, it is almost certainly greater than 6% (15). Even if the calculated MSP1D1 charge was accurate, the resulting POPC charge would be near neutral (þ0.06/lipid, calculated as þ1.9 per 63 lipid molecules).…”

Section: Polyelectrolyte Behavior Of Lipid Nanodiscsmentioning

confidence: 99%

“…In addition, the MSPs allow the migration of the Nanodiscs to be monitored using UV optics. Second, membrane-confined electrophoresis (MCE) has been shown to provide accurate charge measurements on macromolecules in physiological solvents using small quantities of material (7,14,15). In this work, we took advantage of these two advances to obtain the first, to our knowledge, systematic measurements of lipid charge.…”

Section: Introductionmentioning

confidence: 99%

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The Charge Properties of Phospholipid Nanodiscs

Her

Filoti

McLean

et al. 2016

Biophysical Journal

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Phospholipids (PLs) are a major, diverse constituent of cell membranes. PL diversity arises from the nature of the fatty acid chains, as well as the headgroup structure. The headgroup charge is thought to contribute to both the strength and specificity of protein-membrane interactions. Because it has been difficult to measure membrane charge, ascertaining the role charge plays in these interactions has been challenging. Presented here are charge measurements on lipid Nanodiscs at 20 C in 100 mM NaCl, 50 mM Tris, at pH 7.4. Values are also reported for measurements made in the presence of Ca 2þand Mg 2þ as a function of NaCl concentration, pH, and temperature, and in solvents containing other types of cations and anions. Measurements were made for neutral (phosphatidylcholine and phosphatidylethanolamine) and anionic (phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylinositol 4,5-bisphosphate (PIP 2 )) PLs containing palmitoyl-oleoyl and dimyristoyl fatty acid chains. In addition, charge measurements were made on Nanodiscs containing an Escherichia coli lipid extract. The data collected reveal that 1) POPE is anionic and not neutral at pH 7.4; 2) high-anionic-content Nanodiscs exhibit polyelectrolyte behavior; 3) 3 mM Ca 2þ neutralizes a constant fraction of the charge, but not a constant amount of charge, for POPS and POPC Nanodiscs; 4) in contrast to some previous work, POPC only interacts weakly with Ca 2þ ; 5) divalent cations interact with lipids in a lipid-and ion-specific manner for POPA and PIP 2 lipids; and 6) the monovalent anion type has little influence on the lipid charge. These results should help eliminate inconsistencies among data obtained using different techniques, membrane systems, and experimental conditions, and they provide foundational data for developing an accurate view of membranes and membrane-protein interactions.

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Section: Polyelectrolyte Behavior Of Lipid Nanodiscsmentioning

confidence: 99%

Section: Polyelectrolyte Behavior Of Lipid Nanodiscsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Charge Properties of Phospholipid Nanodiscs

Her

Filoti

McLean

et al. 2016

Biophysical Journal

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“…The electrophoretic mobility of BSA in 10 mM Tris + 10 mM NaCl at pH 8.1 was measured using ELS in two different instruments, capillary electrophoresis, and membrane confined electrophoresis [54]. Results obtained by the various methods were in general but not precise agreement, and values of the Debye-Hückel-Henry effective charge calculated from these mobilities were likewise in general but not precise agreement with results calculated from hydrogen ion titration and analysis of amino acid sequence and protein structure.…”

Section: Topics Of Concernmentioning

confidence: 99%

Recent applications of light scattering measurement in the biological and biopharmaceutical sciences

Minton

2016

Analytical Biochemistry

115

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“…We are left in a quandary. However, based on the premise that z p is the charge contained in a hypothetical volume defined by the Stokes radius, Filoti et al proposed that z DHH is an accurate estimate of z P if the Debye-Hückel cloud is considered to be the solvent response to z P (Filoti et al 2015).…”

mentioning

confidence: 99%

Charge matters

Laue

2016

Biophys Rev

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Comparative Study of Analytical Techniques for Determining Protein Charge

Cited by 46 publications

References 27 publications

The Charge Properties of Phospholipid Nanodiscs

The Charge Properties of Phospholipid Nanodiscs

Recent applications of light scattering measurement in the biological and biopharmaceutical sciences

Charge matters

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