Limits...
Spectroscopic and AFM characterization of polypeptide-surface interactions: Controls and lipid quantitative analyses

View Article: PubMed Central - PubMed

ABSTRACT

This article is related to http://dx.doi.org/10.1016/j.bbamem.2017.01.005 (Ø. Strømland, Ø.S. Handegård, M.L. Govasli, H. Wen, Ø. Halskau, 2017) [1]. In protein and polypeptide-membrane interaction studies, negatively charged lipids are often used as they are a known driver for membrane interaction. When using fluorescence spectroscopy and CD as indicators of polypeptide binding and conformational change, respectively, the effect of zwitterionic lipids only should be documented. The present data documents several aspects of how two engineered polypeptides (A-Cage-C and A-Lnk-C) derived from the membrane associating protein alpha-Lactalbumin affects and are affected by the presence of zwitterionic bilayers in the form of vesicles. We here document the behavior or the Cage and Lnk segments with respect to membrane interaction and their residual fold, using intrinsic tryptophan fluorescence assays. This data description also documents the coverage of solid-supported bilayers prepared by spin-coating mica using binary lipid mixes, a necessary step to ensure that AFM is performed on areas that are covered by lipid bilayers when performing experiments. Uncovered patches are detectable by both force curve measurements and height measurements. We tested naked mica׳s ability to cause aggregation as seen by AFM, and found this to be low compared to preparations containing negatively charged lipids. Work with lipids also carries the risk of chemical degradation taking place during vesicles preparation or other handling of the lipids. We therefor use 31P NMR to quantify the head-group content of commonly used commercial extracts before and after a standard protocol for vesicle production is applied.

No MeSH data available.


Related in: MedlinePlus

Association of A-Lnk-C on naked mica imaged by AFM in solution. AFM z-sensor images were acquired of A-Lnk-C, with the indicated concentrations, at pH 7.4 (upper panels) on mica. Subsequently, the pH was lowered to pH 4.5 and images were acquired (lower panels) All images were acquired in tapping mode, and subjected to standard flattening procedures using Gwyddion. Scale bars are 1 µM and 200 nm for the insets, respectively. The height color gradient is 0–15 nm for all images.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384297&req=5

f0045: Association of A-Lnk-C on naked mica imaged by AFM in solution. AFM z-sensor images were acquired of A-Lnk-C, with the indicated concentrations, at pH 7.4 (upper panels) on mica. Subsequently, the pH was lowered to pH 4.5 and images were acquired (lower panels) All images were acquired in tapping mode, and subjected to standard flattening procedures using Gwyddion. Scale bars are 1 µM and 200 nm for the insets, respectively. The height color gradient is 0–15 nm for all images.

Mentions: The size distributions of large unilamellar vesicles (LUVs) prepared by extrusion are presented in (Fig. 1). Solution phase 31P NMR was used to determine the lipid compositions of the LUVs and to elucidate whether modulations in lipid composition occurred during vesicle preparation (Fig. 2). The fold and membrane perturbation capability of the linking segments, Cage-only and Lnk-Only, from the A-Lnk-C and A-Cage-C peptides was investigated by urea denaturation and a Förster Resonance Energy Transfer (FRET) -based membrane leakage assay (Fig. 3, Fig. 4). The tryptophan environment and secondary structure of A-Lnk-C and A-Cage-C in the presence and absence of zwitterionic LUVs is shown in (Fig. 5, Fig. 6). Then, the efficacy of depositing lipid bilayers on mica by spin-coating was elucidated by Atomic Force Microscopy (AFM). The presence or absence of bilayers were assessed by probing the prepared surfaces using the tip of the AFM cantilever (Fig. 7A), and height-profiling of the bilayers (Fig. 7BC). Finally, the aggregation of the polypeptides at neutral and acidic conditions in the presence of mica was investigated by AFM in solution (Fig. 8, Fig. 9).


Spectroscopic and AFM characterization of polypeptide-surface interactions: Controls and lipid quantitative analyses
Association of A-Lnk-C on naked mica imaged by AFM in solution. AFM z-sensor images were acquired of A-Lnk-C, with the indicated concentrations, at pH 7.4 (upper panels) on mica. Subsequently, the pH was lowered to pH 4.5 and images were acquired (lower panels) All images were acquired in tapping mode, and subjected to standard flattening procedures using Gwyddion. Scale bars are 1 µM and 200 nm for the insets, respectively. The height color gradient is 0–15 nm for all images.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384297&req=5

f0045: Association of A-Lnk-C on naked mica imaged by AFM in solution. AFM z-sensor images were acquired of A-Lnk-C, with the indicated concentrations, at pH 7.4 (upper panels) on mica. Subsequently, the pH was lowered to pH 4.5 and images were acquired (lower panels) All images were acquired in tapping mode, and subjected to standard flattening procedures using Gwyddion. Scale bars are 1 µM and 200 nm for the insets, respectively. The height color gradient is 0–15 nm for all images.
Mentions: The size distributions of large unilamellar vesicles (LUVs) prepared by extrusion are presented in (Fig. 1). Solution phase 31P NMR was used to determine the lipid compositions of the LUVs and to elucidate whether modulations in lipid composition occurred during vesicle preparation (Fig. 2). The fold and membrane perturbation capability of the linking segments, Cage-only and Lnk-Only, from the A-Lnk-C and A-Cage-C peptides was investigated by urea denaturation and a Förster Resonance Energy Transfer (FRET) -based membrane leakage assay (Fig. 3, Fig. 4). The tryptophan environment and secondary structure of A-Lnk-C and A-Cage-C in the presence and absence of zwitterionic LUVs is shown in (Fig. 5, Fig. 6). Then, the efficacy of depositing lipid bilayers on mica by spin-coating was elucidated by Atomic Force Microscopy (AFM). The presence or absence of bilayers were assessed by probing the prepared surfaces using the tip of the AFM cantilever (Fig. 7A), and height-profiling of the bilayers (Fig. 7BC). Finally, the aggregation of the polypeptides at neutral and acidic conditions in the presence of mica was investigated by AFM in solution (Fig. 8, Fig. 9).

View Article: PubMed Central - PubMed

ABSTRACT

This article is related to http://dx.doi.org/10.1016/j.bbamem.2017.01.005 (Ø. Strømland, Ø.S. Handegård, M.L. Govasli, H. Wen, Ø. Halskau, 2017) [1]. In protein and polypeptide-membrane interaction studies, negatively charged lipids are often used as they are a known driver for membrane interaction. When using fluorescence spectroscopy and CD as indicators of polypeptide binding and conformational change, respectively, the effect of zwitterionic lipids only should be documented. The present data documents several aspects of how two engineered polypeptides (A-Cage-C and A-Lnk-C) derived from the membrane associating protein alpha-Lactalbumin affects and are affected by the presence of zwitterionic bilayers in the form of vesicles. We here document the behavior or the Cage and Lnk segments with respect to membrane interaction and their residual fold, using intrinsic tryptophan fluorescence assays. This data description also documents the coverage of solid-supported bilayers prepared by spin-coating mica using binary lipid mixes, a necessary step to ensure that AFM is performed on areas that are covered by lipid bilayers when performing experiments. Uncovered patches are detectable by both force curve measurements and height measurements. We tested naked mica׳s ability to cause aggregation as seen by AFM, and found this to be low compared to preparations containing negatively charged lipids. Work with lipids also carries the risk of chemical degradation taking place during vesicles preparation or other handling of the lipids. We therefor use 31P NMR to quantify the head-group content of commonly used commercial extracts before and after a standard protocol for vesicle production is applied.

No MeSH data available.


Related in: MedlinePlus