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The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues.

Horner A, Zocher F, Preiner J, Ollinger N, Siligan C, Akimov SA, Pohl P - Sci Adv (2015)

Bottom Line: We show that both the p f of those channels and the diffusion coefficient of the single-file waters within them are determined by the number N H of residues in the channel wall that may form a hydrogen bond with the single-file waters.The logarithmic dependence of water diffusivity on N H is in line with the multiplicity of binding options at higher N H densities.We obtained high-precision p f values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Johannes Kepler University Linz, Institute of Biophysics, Gruberstr. 40, 4020 Linz, Austria.

ABSTRACT

Channel geometry governs the unitary osmotic water channel permeability, p f, according to classical hydrodynamics. Yet, p f varies by several orders of magnitude for membrane channels with a constriction zone that is one water molecule in width and four to eight molecules in length. We show that both the p f of those channels and the diffusion coefficient of the single-file waters within them are determined by the number N H of residues in the channel wall that may form a hydrogen bond with the single-file waters. The logarithmic dependence of water diffusivity on N H is in line with the multiplicity of binding options at higher N H densities. We obtained high-precision p f values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

No MeSH data available.


Related in: MedlinePlus

Determination of reconstitution efficiency(A) FCS autocorrelation curves allowed us to obtain the number of (i) vesicles labeled with 0.004% (w/w) N-(lissamine-rhodamine-sulfonyl)phosphatidylethanolamine (sandy brown), (ii) AQP1-YFP–containing vesicles (purple), (iii) AQP1-YFP oligomers containing micelles that formed upon vesicle dissolution in mild detergent (dashed purple), and (iv) AQP1-YFP monomer containing micelles that formed upon further dissolution in harsh detergent (dotted purple) per confocal volume. The buffer (pH 7.4) contained 100 mM NaCl, 20 mM Mops, and a protease inhibitor. (B and C) AFM imaging of solid-supported lipid bilayers that were prepared from AQPZ proteoliposomes (B) or empty vesicles (investigated area: 14 × 400 × 400nm2) (C) resulted in histograms of height values (n = 50). (B) Inset: The high-resolution raw data allowed differentiation of the extracellular and cytoplasmic AQPZ surfaces. (C) The density of unspecified features (0.218 per vesicle) served to correct the protein count (see also fig. S1). (D) Comparison of both the absolute AFM and FCS counts of AQPZ tetramers per liposome (at three different concentrations) (upper panel) and their ratio (middle panel). Average ratio of AFM and FCS counts per liposome for AQPZ, GlpF, and AQP1 oligomers (lower panel: compare also eqs. S9 and S10).
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Figure 1: Determination of reconstitution efficiency(A) FCS autocorrelation curves allowed us to obtain the number of (i) vesicles labeled with 0.004% (w/w) N-(lissamine-rhodamine-sulfonyl)phosphatidylethanolamine (sandy brown), (ii) AQP1-YFP–containing vesicles (purple), (iii) AQP1-YFP oligomers containing micelles that formed upon vesicle dissolution in mild detergent (dashed purple), and (iv) AQP1-YFP monomer containing micelles that formed upon further dissolution in harsh detergent (dotted purple) per confocal volume. The buffer (pH 7.4) contained 100 mM NaCl, 20 mM Mops, and a protease inhibitor. (B and C) AFM imaging of solid-supported lipid bilayers that were prepared from AQPZ proteoliposomes (B) or empty vesicles (investigated area: 14 × 400 × 400nm2) (C) resulted in histograms of height values (n = 50). (B) Inset: The high-resolution raw data allowed differentiation of the extracellular and cytoplasmic AQPZ surfaces. (C) The density of unspecified features (0.218 per vesicle) served to correct the protein count (see also fig. S1). (D) Comparison of both the absolute AFM and FCS counts of AQPZ tetramers per liposome (at three different concentrations) (upper panel) and their ratio (middle panel). Average ratio of AFM and FCS counts per liposome for AQPZ, GlpF, and AQP1 oligomers (lower panel: compare also eqs. S9 and S10).

Mentions: We determined the number n of aquaporin monomers per proteoliposome by FCS as previously described (25) and as exemplified in Fig. 1A (see also fig. S5). Depending on the preparation, n varied between 1 and 30. The reconstitution efficiency varied between 10 and 50% (fig. S6). At very low concentrations, AQP1 and GlpF reconstituted as functional monomers, dimers, or trimers. To enable comparison with AFM measurements, we calculated the expected number NO,FCS of oligomers per proteoliposome by assuming (i) a Poisson distribution of functional units among the proteoliposomes and (ii) that all units within one vesicle assembled into the smallest number of oligomers possible.


The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues.

Horner A, Zocher F, Preiner J, Ollinger N, Siligan C, Akimov SA, Pohl P - Sci Adv (2015)

Determination of reconstitution efficiency(A) FCS autocorrelation curves allowed us to obtain the number of (i) vesicles labeled with 0.004% (w/w) N-(lissamine-rhodamine-sulfonyl)phosphatidylethanolamine (sandy brown), (ii) AQP1-YFP–containing vesicles (purple), (iii) AQP1-YFP oligomers containing micelles that formed upon vesicle dissolution in mild detergent (dashed purple), and (iv) AQP1-YFP monomer containing micelles that formed upon further dissolution in harsh detergent (dotted purple) per confocal volume. The buffer (pH 7.4) contained 100 mM NaCl, 20 mM Mops, and a protease inhibitor. (B and C) AFM imaging of solid-supported lipid bilayers that were prepared from AQPZ proteoliposomes (B) or empty vesicles (investigated area: 14 × 400 × 400nm2) (C) resulted in histograms of height values (n = 50). (B) Inset: The high-resolution raw data allowed differentiation of the extracellular and cytoplasmic AQPZ surfaces. (C) The density of unspecified features (0.218 per vesicle) served to correct the protein count (see also fig. S1). (D) Comparison of both the absolute AFM and FCS counts of AQPZ tetramers per liposome (at three different concentrations) (upper panel) and their ratio (middle panel). Average ratio of AFM and FCS counts per liposome for AQPZ, GlpF, and AQP1 oligomers (lower panel: compare also eqs. S9 and S10).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496530&req=5

Figure 1: Determination of reconstitution efficiency(A) FCS autocorrelation curves allowed us to obtain the number of (i) vesicles labeled with 0.004% (w/w) N-(lissamine-rhodamine-sulfonyl)phosphatidylethanolamine (sandy brown), (ii) AQP1-YFP–containing vesicles (purple), (iii) AQP1-YFP oligomers containing micelles that formed upon vesicle dissolution in mild detergent (dashed purple), and (iv) AQP1-YFP monomer containing micelles that formed upon further dissolution in harsh detergent (dotted purple) per confocal volume. The buffer (pH 7.4) contained 100 mM NaCl, 20 mM Mops, and a protease inhibitor. (B and C) AFM imaging of solid-supported lipid bilayers that were prepared from AQPZ proteoliposomes (B) or empty vesicles (investigated area: 14 × 400 × 400nm2) (C) resulted in histograms of height values (n = 50). (B) Inset: The high-resolution raw data allowed differentiation of the extracellular and cytoplasmic AQPZ surfaces. (C) The density of unspecified features (0.218 per vesicle) served to correct the protein count (see also fig. S1). (D) Comparison of both the absolute AFM and FCS counts of AQPZ tetramers per liposome (at three different concentrations) (upper panel) and their ratio (middle panel). Average ratio of AFM and FCS counts per liposome for AQPZ, GlpF, and AQP1 oligomers (lower panel: compare also eqs. S9 and S10).
Mentions: We determined the number n of aquaporin monomers per proteoliposome by FCS as previously described (25) and as exemplified in Fig. 1A (see also fig. S5). Depending on the preparation, n varied between 1 and 30. The reconstitution efficiency varied between 10 and 50% (fig. S6). At very low concentrations, AQP1 and GlpF reconstituted as functional monomers, dimers, or trimers. To enable comparison with AFM measurements, we calculated the expected number NO,FCS of oligomers per proteoliposome by assuming (i) a Poisson distribution of functional units among the proteoliposomes and (ii) that all units within one vesicle assembled into the smallest number of oligomers possible.

Bottom Line: We show that both the p f of those channels and the diffusion coefficient of the single-file waters within them are determined by the number N H of residues in the channel wall that may form a hydrogen bond with the single-file waters.The logarithmic dependence of water diffusivity on N H is in line with the multiplicity of binding options at higher N H densities.We obtained high-precision p f values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Johannes Kepler University Linz, Institute of Biophysics, Gruberstr. 40, 4020 Linz, Austria.

ABSTRACT

Channel geometry governs the unitary osmotic water channel permeability, p f, according to classical hydrodynamics. Yet, p f varies by several orders of magnitude for membrane channels with a constriction zone that is one water molecule in width and four to eight molecules in length. We show that both the p f of those channels and the diffusion coefficient of the single-file waters within them are determined by the number N H of residues in the channel wall that may form a hydrogen bond with the single-file waters. The logarithmic dependence of water diffusivity on N H is in line with the multiplicity of binding options at higher N H densities. We obtained high-precision p f values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

No MeSH data available.


Related in: MedlinePlus