Trends in Thermostability Provide Information on the Nature of Substrate, Inhibitor, and Lipid Interactions with Mitochondrial Carriers

Crichton, Paul G.; Lee, Yang; Ruprecht, Jonathan J.; Cerson, Elizabeth; Thangaratnarajah, Chancievan; King, Martin S.; Kunji, Edmund R. S.

doi:10.1074/jbc.m114.616607

Cited by 79 publications

(139 citation statements)

References 60 publications

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“…to form a blue fluorescent adduct [97]. In the procedure, the temperature of purified protein samples is increased from 25 to 90 °C while protein unfolding is monitored with CPM, as buried cysteine residues become solvent exposed due to thermal denaturation of the protein [98]. B Representative unfolding profiles of uninhibited AAC (black line), CATR--inhibited AAC (blue line) and BKA--inhibited AAC (red line) in detergent [96].…”

Section: Resultsmentioning

confidence: 99%

“…The probe was used in an assay in the Stevens lab to test the relative stability of membrane proteins to aid crystallisation trials [97], but we have adapted the assay and developed it as a powerful tool to obtain key biological information. In the procedure, the temperature of purified protein samples is increased from 25 to 90 °C, buried cysteine residues become solvent exposed due to thermal denaturation of the protein and an unfolding curve is obtained through fluorescence measurements [98]. The peak in the derivative of the unfolding curve, the "melting temperature", provides a relative measure of protein stability.…”

Section: Figure 12 Proposed Transport Mechanism Of the Mitochondrialmentioning

confidence: 99%

“…The peak in the derivative of the unfolding curve, the "melting temperature", provides a relative measure of protein stability. We have used the assay to provide information on detergent, lipid, substrate and inhibitor interactions with various mitochondrial carriers and uncoupling proteins [78,96,98].…”

Section: Figure 12 Proposed Transport Mechanism Of the Mitochondrialmentioning

confidence: 99%

See 2 more Smart Citations

The transport mechanism of the mitochondrial ADP/ATP carrier

Kunji

Aleksandrova

King

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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124

141

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The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, providing key steps in oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α--helices linked with a loop and short α--helix on the matrix side. The transporter cycles between a cytoplasmic and matrix conformation in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic conformation, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix conformation. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. The simultaneous 2 rotation of three domains around a central translocation pathway constitutes a unique mechanism among transport proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Figure 12 Proposed Transport Mechanism Of the Mitochondrialmentioning

confidence: 99%

Section: Figure 12 Proposed Transport Mechanism Of the Mitochondrialmentioning

confidence: 99%

See 1 more Smart Citation

The transport mechanism of the mitochondrial ADP/ATP carrier

Kunji

Aleksandrova

King

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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124

141

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“…CPM reacts with protein-buried cysteine residues as they become solvent exposed due to thermal denaturation to give a fluorescent adduct (41,42). In tests, ovine UCP1 exhibited a background fluorescence (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Crucially, these motifs are present in UCP1 (although the glycine at position 248 is replaced by serine, also a helix breaker), indicating that UCP1 most likely binds cardiolipin in a similar manner as the mitochondrial ADP/ATP carrier. The observed stabilizing effect of cardiolipin and other lipids here is most likely achieved by increasing the size of the supporting detergent micelle (42), which, in turn, may impact on the behavior of the tightly bound species.…”

Section: Discussionmentioning

confidence: 99%

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Lee

Willers

Kunji

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein.Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria.

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Molecular Dynamics Simulations of the Elusive Matrix‐Open State of Mitochondrial ADP/ATP Carrier

Škulj

Brkljača

Vazdar

2020

Israel Journal of Chemistry

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We studied and compared in detail an elusive matrix open (m‐state) and cytoplasmic open (c‐state) state of ADP/ATP carrier (AAC) protein embedded in the DOPC bilayer by microsecond molecular dynamics (MD) simulations. We analyzed both states with and without cardiolipin (CDL) molecules, with a special emphasis on the recently obtained crystallographic structure of the AAC m‐state. The obtained results show that both states of the protein are stable in the DOPC bilayer and impermeable to water. In comparison with the c‐state of AAC, the m‐state is more dynamic, but at the same time possesses a larger occluded area in the protein cavity. Both states of the protein are less flexible in simulations when CDL molecules are present, which is especially visible for the m‐state. Finally, the analysis of the protein conformational changes during MD simulations shows that protein parts at the protein/lipid boundaries are prone to larger conformational changes in contrast to central region of the protein embedded in the bilayer core, thus further supporting the cycling mechanism suggested for ADP/ATP exchange by AAC.

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Trends in Thermostability Provide Information on the Nature of Substrate, Inhibitor, and Lipid Interactions with Mitochondrial Carriers

Cited by 79 publications

References 60 publications

The transport mechanism of the mitochondrial ADP/ATP carrier

The transport mechanism of the mitochondrial ADP/ATP carrier

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Molecular Dynamics Simulations of the Elusive Matrix‐Open State of Mitochondrial ADP/ATP Carrier

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