Unfolding study of a trimeric membrane protein AcrB

Cui, Ye; Wang, Zhaoshuai; Lu, Wei; Wei, Yinan

doi:10.1002/pro.2471

Cited by 6 publications

(8 citation statements)

References 42 publications

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“…However, we have previously found that the presence of intersubunit disulfide bonds, which strengthen AcrB trimer stability by covalently linking the three subunits, has little effect on the transition point of AcrB unfolding monitored using fluorescence intensity. 25 The fluorescence intensity change indicates the exposure of previously hidden aromatic residues, which results from the disruption of the hydrophobic core or dissociation of subunits. There are two potential reasons that may lead to this observation.…”

Section: Resultsmentioning

confidence: 99%

“…We also found that the change of the fluorescence signal is largely contributed by aromatic residues in the soluble domain. 25 The unfolding of the transmembrane domain may not generate a detectable fluorescence signal change, as the change of microenvironment for Trp residues in the transmembrane domain upon unfolding is not likely to be as dramatic as for Trp residues in the soluble domain.…”

Section: Resultsmentioning

confidence: 99%

“… 24 SDS-induced unfolding was monitored using fluorescence spectroscopy as described previously. 25 Briefly, unfolding of AcrB was initiated by titrating with a 0.25% (w/v) SDS solution. A fluorescence emission spectrum ranging from 300 to 400 nm was recorded at each titration point on a LS-55 fluorescence spectrometer (PerkinElmer, Inc., Waltham, MA) with an excitation wavelength of 280 nm.…”

Section: Methodsmentioning

confidence: 99%

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Correlation between AcrB Trimer Association Affinity and Efflux Activity

Cui

Wang

et al. 2014

Biochemistry

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The majority of membrane proteins function as oligomers. However, it remains largely unclear how the oligomer stability of protein complexes correlates with their function. Understanding the relationship between oligomer stability and activity is essential to protein research and to virtually all cellular processes that depend on the function of protein complexes. Proteins make lasting or transient interactions as they perform their functions. Obligate oligomeric proteins exist and function exclusively at a specific oligomeric state. Although oligomerization is clearly critical for such proteins to function, a direct correlation between oligomer affinity and biological activity has not yet been reported. Here, we used an obligate trimeric membrane transporter protein, AcrB, as a model to investigate the correlation between its relative trimer affinity and efflux activity. AcrB is a component of the major multidrug efflux system in Escherichia coli. We created six AcrB constructs with mutations at the transmembrane intersubunit interface, and we determined their activities using both a drug susceptibility assay and an ethidium bromide accumulation assay. The relative trimer affinities of these mutants in detergent micelles were obtained using blue native polyacrylamide gel electrophoresis. A correlation between the relative trimer affinity and substrate efflux activity was observed, in which a threshold trimer stability was required to maintain efflux activity. The trimer affinity of the wild-type protein was approximately 3 kcal/mol more stable than the threshold value. Once the threshold was reached, an additional increase of stability in the range observed had no observable effect on protein activity.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Correlation between AcrB Trimer Association Affinity and Efflux Activity

Cui

Wang

et al. 2014

Biochemistry

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“…Although many membrane proteins have been successfully refolded after incubation in mild denaturants such as SDS (41), it is less common for a-helical membrane proteins to be successfully refolded to an active state after substantial unfolding (42). In some cases, native-like secondary structure can be restored, although reassociation of different subunits seems to be a limiting factor, as observed with the multidrug transporter, AcrB (43). Both HiGlpG and PsAarA exist as dimers in DDM (44); indeed, we observe a dimeric state for both HiGlpG and PsAarA upon refolding.…”

Section: Tertiary Structure and Activity Upon Refoldingmentioning

confidence: 99%

Reversible Unfolding of Rhomboid Intramembrane Proteases

Panigrahi

Arutyunova

Panwar

et al. 2016

Biophysical Journal

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Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.

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“…Interestingly, almost all suggest that oligomerization is a cooperative rather than sequential process. In unfolding and refolding studies of the tetrameric KscA using triflouroethanol (Valiyaveetil et al , 2002; Barrera et al , 2008) and of the trimeric AcrB using SDS and urea (Ye et al , 2014a), no intermediates were observed. Unfolding and refolding studies of AcrB and the ABC transporter BtuCD even suggested that oligomerization might be coupled with folding.…”

Section: Introductionmentioning

confidence: 99%

How physical forces drive the process of helical membrane protein folding

Corin

Bowie

2022

EMBO Reports

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Protein folding is a fundamental process of life with important implications throughout biology. Indeed, tens of thousands of mutations have been associated with diseases, and most of these mutations are believed to affect protein folding rather than function. Correct folding is also a key element of design. These factors have motivated decades of research on protein folding. Unfortunately, knowledge of membrane protein folding lags that of soluble proteins. This gap is partly caused by the greater technical challenges associated with membrane protein studies, but also because of additional complexities. While soluble proteins fold in a homogenous water environment, membrane proteins fold in a setting that ranges from bulk water to highly charged to apolar. Thus, the forces that drive folding vary in different regions of the protein, and this complexity needs to be incorporated into our understanding of the folding process. Here, we review our understanding of membrane protein folding biophysics. Despite the greater challenge, better model systems and new experimental techniques are starting to unravel the forces and pathways in membrane protein folding.

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Unfolding study of a trimeric membrane protein AcrB

Cited by 6 publications

References 42 publications

Correlation between AcrB Trimer Association Affinity and Efflux Activity

Correlation between AcrB Trimer Association Affinity and Efflux Activity

Reversible Unfolding of Rhomboid Intramembrane Proteases

How physical forces drive the process of helical membrane protein folding

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