Abstract:Evasion from programmed cell death (apoptosis) is the main hallmark of cancer and a major cause of resistance to therapy. Many tumors simply ensure survival by over-expressing the cell-protecting (anti-apoptotic) Bcl-2 membrane protein involved in apoptotic regulation. However, the molecular mechanism by which Bcl-2 protein in its mitochondrial outer membrane location protects cells remains elusive due to the absence of structural insight; and current strategies to therapeutically interfere with these Bcl-2 se… Show more
“…Comparison with previous solid-state NMR and neutron reflectometry studies on Bcl-2 embedded in lipid bilayers ( 12 ) and refolding experiments into detergent systems using a soluble Bcl-2 variant without its TM domain ( 24 ) clearly shows that the micellar environment used here reflects well the Bcl-2’s globular fold into a compact membrane-like state. This is also visible by most residues buried and motionally restricted in a micellar environment, whereas the FLD region is flexible and solvent exposed, as seen here in Figs.…”
Section: Resultsmentioning
confidence: 52%
“…There are also small effects in the TM region containing the C-terminus (G227 to G237) as shown by affected 1 H resonances. Except for glycine 5 in the N-terminus region, the other two glycines G8 and G27 near or inherent to neighboring BH4 ( 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ) domain show smaller CSPs. As expected, the intrinsically disordered FLD region (33–90 aa) shows no involvement in the binding in this region (G41 to G83), whereas at its connection to the membrane-active BH4 domain of Bcl-2 ( 33 ), variations are visible for the glycine residues there.…”
Section: Resultsmentioning
confidence: 99%
“…All those similar K D values reveal moderate binding affinities across the entire protein. The glycine-specific CSP patterns and varying affinities indicate a simultaneous occurrence of binding that occurs, which nevertheless display a complex behavior, presumably reflecting the domain organization of Bcl-2 and the occurring changes in the membrane-mimicking environment ( 24 ). This complexity is also seen for glycines corresponding to the binding domain (G101, G141, G145).…”
Section: Resultsmentioning
confidence: 99%
“…The Bcl-2 wild-type protein as well as the truncated variants Bcl-2 ΔN(1–82), Bcl-2 ΔTM (208–239), Bcl-2 ΔN (1–82) ΔTM (208–239), and Bcl-2 ΔC(93–239) were expressed in 15 NH 4 Cl or 15 NH 4 Cl/ 13 C glucose-enriched M9 minimal media and purified according to the previously described procedure ( 24 ). For Bcl-2/Bax interaction studies, the 36-mer Bax-BH3 peptide (Ac-QPPQDASTKKLSECLRRIGDELDSNMELQRMIADVD-NH 2 ) from Mus musculus ( 19 ) was purchased from GenScript (Leiden, the Netherlands) and dissolved as a 20 mM stock using a buffer consisting of 5 mM DPC micelles, 20 mM NaPi, 20 mM NaCl, and 2 mM TCEP at pH 6.0.…”
Section: Methodsmentioning
confidence: 99%
“…Only recently, we obtained a first direct insight into the location of Bcl-2 as an embedded membrane protein by combined neutron reflectometry and nuclear magnetic resonance (NMR) studies ( 12 ). To unravel the molecular basis of the recognition of Bax via its death domain by the Bcl-2 protein and to understand the respective response of the entire Bcl-2 protein and especially its binding groove interface, we here combined liquid-state NMR methods with isotope-labeled, full-length, functional human Bcl-2 protein in a membrane-mimicking micellar environment, a strategy we recently developed ( 24 ). By identifying all individual glycine residues distributed across the entire Bcl-2 protein in the corresponding NMR spectra, we were able to trace each glycine residue and protein domain individually to monitor their specific response upon an increasing presence of a Bax-derived 36-mer BH3 domain peptide (residues 49–84 in the Bax sequence ( 19 )).…”
“…Comparison with previous solid-state NMR and neutron reflectometry studies on Bcl-2 embedded in lipid bilayers ( 12 ) and refolding experiments into detergent systems using a soluble Bcl-2 variant without its TM domain ( 24 ) clearly shows that the micellar environment used here reflects well the Bcl-2’s globular fold into a compact membrane-like state. This is also visible by most residues buried and motionally restricted in a micellar environment, whereas the FLD region is flexible and solvent exposed, as seen here in Figs.…”
Section: Resultsmentioning
confidence: 52%
“…There are also small effects in the TM region containing the C-terminus (G227 to G237) as shown by affected 1 H resonances. Except for glycine 5 in the N-terminus region, the other two glycines G8 and G27 near or inherent to neighboring BH4 ( 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ) domain show smaller CSPs. As expected, the intrinsically disordered FLD region (33–90 aa) shows no involvement in the binding in this region (G41 to G83), whereas at its connection to the membrane-active BH4 domain of Bcl-2 ( 33 ), variations are visible for the glycine residues there.…”
Section: Resultsmentioning
confidence: 99%
“…All those similar K D values reveal moderate binding affinities across the entire protein. The glycine-specific CSP patterns and varying affinities indicate a simultaneous occurrence of binding that occurs, which nevertheless display a complex behavior, presumably reflecting the domain organization of Bcl-2 and the occurring changes in the membrane-mimicking environment ( 24 ). This complexity is also seen for glycines corresponding to the binding domain (G101, G141, G145).…”
Section: Resultsmentioning
confidence: 99%
“…The Bcl-2 wild-type protein as well as the truncated variants Bcl-2 ΔN(1–82), Bcl-2 ΔTM (208–239), Bcl-2 ΔN (1–82) ΔTM (208–239), and Bcl-2 ΔC(93–239) were expressed in 15 NH 4 Cl or 15 NH 4 Cl/ 13 C glucose-enriched M9 minimal media and purified according to the previously described procedure ( 24 ). For Bcl-2/Bax interaction studies, the 36-mer Bax-BH3 peptide (Ac-QPPQDASTKKLSECLRRIGDELDSNMELQRMIADVD-NH 2 ) from Mus musculus ( 19 ) was purchased from GenScript (Leiden, the Netherlands) and dissolved as a 20 mM stock using a buffer consisting of 5 mM DPC micelles, 20 mM NaPi, 20 mM NaCl, and 2 mM TCEP at pH 6.0.…”
Section: Methodsmentioning
confidence: 99%
“…Only recently, we obtained a first direct insight into the location of Bcl-2 as an embedded membrane protein by combined neutron reflectometry and nuclear magnetic resonance (NMR) studies ( 12 ). To unravel the molecular basis of the recognition of Bax via its death domain by the Bcl-2 protein and to understand the respective response of the entire Bcl-2 protein and especially its binding groove interface, we here combined liquid-state NMR methods with isotope-labeled, full-length, functional human Bcl-2 protein in a membrane-mimicking micellar environment, a strategy we recently developed ( 24 ). By identifying all individual glycine residues distributed across the entire Bcl-2 protein in the corresponding NMR spectra, we were able to trace each glycine residue and protein domain individually to monitor their specific response upon an increasing presence of a Bax-derived 36-mer BH3 domain peptide (residues 49–84 in the Bax sequence ( 19 )).…”
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