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. 2007 Jan 15;401(2):437-45.
doi: 10.1042/BJ20061046.

Interaction of mammalian Hsp22 with lipid membranes

Tirumala Kumar Chowdary  1 , Bakthisaran Raman, Tangirala Ramakrishna, Ch Mohan Rao
Affiliations

Interaction of mammalian Hsp22 with lipid membranes

Tirumala Kumar Chowdary et al. Biochem J. .

Abstract

Hsp22/HspB8 is a member of the small heat-shock protein family, whose function is not yet completely understood. Our immunolocalization studies in a human neuroblastoma cell line, SK-N-SH, using confocal microscopy show that a significant fraction of Hsp22 is localized to the plasma membrane. We therefore investigated its interactions with lipid vesicles in vitro. Intrinsic tryptophan fluorescence is quenched in the presence of lipid vesicles derived from either bovine brain lipid extract or purified lipids. Time-resolved fluorescence studies show a decrease in the lifetimes of the tryptophan residues. Both of these results indicate burial of some tryptophan residues of Hsp22 upon interaction with lipid vesicles. Membrane interactions also lead to increase in fluorescence polarization of Hsp22. Gel-filtration chromatography shows that Hsp22 binds stably with lipid vesicles; the extent of binding depends on the nature of the lipid. Hsp22 binds more strongly to vesicles made of lipids containing a phosphatidic acid, phosphatidylinositol or phosphatidylserine headgroup (known to be present in the inner leaflet of plasma membrane) compared with lipid vesicles made of a phosphatidylcholine head-group alone. Far-UV CD spectra reveal conformational changes upon binding to the lipid vesicles or in membrane-mimetic solvent, trifluoroethanol. Thus our fluorescence, CD and gel-filtration studies show that Hsp22 interacts with membrane and this interaction leads to stable binding and conformational changes. The present study therefore clearly demonstrates that Hsp22 exhibits potential membrane interaction that may play an important role in its cellular functions.

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Figures

Figure 1
Figure 1. Immunolocalization of Hsp22 in SK-N-SH cells
(A) Immunolocalization of endogenous Hsp22. (B and C) Immunolocalization of ectopically expressed FLAG-tagged Hsp22. Cells were permeabilized either by acetone treatment (B) or by 0.1% Triton X-100 treatment (C). (D) The transiently transfected cells were treated with 1% Triton X-100 for 20 min after fixation to determine the effect of membrane disruption on the localization of Hsp22. Cells were probed with either polyclonal antibody against Hsp22 (A) or monoclonal antibodies against the FLAG epitope of the transiently expressed FLAG-tagged Hsp22 (B–D) and DiIC16, a membrane-specific dye (all panels). Nuclei were stained with DAPI. Regions of membrane of the cells exhibiting co-localization of Hsp22 and DiIC16 are shown in the merge panel. The cut mask panel shows the co-localized areas masking the rest of the image. This cut mask image is created using the manufacturer-provided (LSM 510 meta, Zeiss) software. All of the images were processed under identical conditions.
Figure 2
Figure 2. Quenching of the tryptophan fluorescence of Hsp22 by lipid vesicles
(A) Intrinsic fluorescence of Hsp22 in the absence and the presence of increasing concentrations of SUVs prepared from BLE FrI. Fluorescence spectra of Hsp22 (0.2 mg/ml) incubated with lipid SUVs at different indicated SUV/protein ratios (w/w) are shown. Excitation wavelength was 295 nm, and excitation and emission band passes were set at 5 nm. (B) Extent of quenching [(F0−F)/F0, where F0 and F are the fluorescence intensities at 340 nm in the absence and in the presence of lipid vesicles respectively] of Hsp22 fluorescence upon interaction with lipid SUVs of BLE FrI (○), BLE FrIII (●) and PC/PA (□) is plotted against different lipid/Hsp22 ratios (w/w). (C) Intrinsic fluorescence of β-lactoglobulin (0.1 mg/ml) in the absence (solid line) and in the presence (broken line) of SUVs (0.5 mg/ml) prepared from BLE FrI. Excitation wavelength was 295 nm, and excitation and emission band passes were set at 5 nm.
Figure 3
Figure 3. Time-resolved tryptophan fluorescence intensity decay of Hsp22
Excitation source was a 294 nm NanoLED pulsed laser, and emission was monitored at 340 nm. Grey circles, fluorescence decay; open black circles, prompt. The experimental fluorescence decay data were fitted assuming a three-exponential decay. Chi. sq, χ2. The lower panel shows the weighted residuals. The calculated fluorescence lifetimes are listed in Table 1.
Figure 4
Figure 4. Effect of lipid association on fluorescence polarization of Hsp22
Fluorescence polarization values of the samples of Hsp22 (0.1 mg/ml) in the absence and the presence of various SUVs at a lipid/protein ratio of 5:1 (w/w) are shown. Results are means±S.E.M. for three independent measurements.
Figure 5
Figure 5. Binding of Hsp22 to lipid SUVs shown by gel-filtration chromatography
(A) Elution profile of Hsp22 alone monitored by fluorescence intensity at 340 nm. (B) Elution profile of a mixture of Hsp22 and PC/PA (1:5, w/w) (◆). The elution of protein was detected by its fluorescence at 340 nm upon excitation at 295 nm. Under this excitation and emission wavelength, the elution of SUVs alone (■) could not be detected, indicating that the fluorescence seen in the mixture is due to protein alone. Inset: Coomassie Blue R250-stained SDS/polyacrylamide gel showing the band corresponding to Hsp22 in the eluted fractions. Upper panel: Hsp22 alone; lower panel: Hsp22+PC/PA. (C) Elution profiles of SUVs in the mixture of Hsp22 and PC/PA (1:5, w/w) monitored using the extrinsic fluorescence probe, DPH (10 μM), added to the fractions. DPH partitions into the membrane bilayer, leading to increase in its fluorescence intensity. It does not yield significant fluorescence in the presence of protein alone. Samples were excited at 358 nm and emission was monitored at 430 nm. (D) Elution profiles of β-lactoglobulin (0.5 mg/ml) in the absence (○) and in the presence of SUVs (2.5 mg/ml) (●). AU, arbitrary units.
Figure 6
Figure 6. Far-UV CD spectra of Hsp22 in the absence or the presence of lipid SUVs
(A) Far-UV CD spectra of Hsp22 alone (○), or in the presence of lipid/protein (5:1, w/w), BLE FrI (+) or BLE FrIII (●). (B) Far-UV CD spectra of Hsp22 alone (○), or in the presence of lipid/protein (5:1, w/w) of PC/PA (+) or PC/PI (●). (C) Far-UV CD spectra of Hsp22 in buffer alone (○) and in 40% trifluoroethanol (△).
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