Analyzing Oligomerization of Individual Transmembrane Helices and of Entire Membrane Proteins in E. coli: A Hitchhiker’s Guide to GALLEX

Cymer, Florian; Sanders, Charles R.; Schneider, Dirk

doi:10.1007/978-1-62703-065-6_16

Cited by 9 publications

(10 citation statements)

References 28 publications

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“…The GALLEX-assay was performed as described in detail recently [35][36][37]. To introduce single and multiple point mutations into the psbF gene, the Quickchange mutagenesis kit was used according to the manufacturer's instructions (Stratagene).…”

Section: Methodssupporting

confidence: 45%

“…Plasmids coding for the psbF gene fused to the LexA DNA-binding domain and to the MalE domain were constructed as described [31]. The GALLEX-assay was performed as described in detail recently [35][36][37]. To introduce single and multiple point mutations into the psbF gene, the Quickchange mutagenesis kit was used according to the manufacturer's instructions (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Six amino acids define a minimal dimerization sequence and stabilize a transmembrane helix dimer by close packing and hydrogen bonding

Weber

Schneider

2013

FEBS Letters

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Edited by Richard CogdellKeywords: Cytochrome b 559 Dimerization motif Transmembrane Helix-helix interaction Sequence context a b s t r a c t Distinct amino acid sequences have been described to mediate oligomerization of transmembrane a-helices. However, as the sequence context is crucial to determine specificity in transmembrane helix-helix interaction, the question arises how small a sequence can be without losing specificity. In the present analysis, six amino acids have been identified in the PsbF transmembrane helix dimer, which form the contact region of two interacting helices and are directly involved in helix-helix interactions. However, individual amino acids within the complex sequence pattern only together ensure sequence specificity of the analyzed transmembrane helix-helix interactions by mediating close packing and inter-helical hydrogen bonding. Structured summary of protein interactions:psbF and psbF physically interact by lex-a dimerization assay (View Interaction: 1,2,3,4,5)

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Section: Methodssupporting

confidence: 45%

Section: Methodsmentioning

confidence: 99%

Six amino acids define a minimal dimerization sequence and stabilize a transmembrane helix dimer by close packing and hydrogen bonding

Weber

Schneider

2013

FEBS Letters

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“…Nevertheless, it is conceivable that the GxxxG motif of the TLR2 and TLR6 TMDs move in a piston mechanism (for an explanation on the piston motion, see Ref. [24]). This motion could explain how these motifs are important for the TLR function although they are not located parallel to each other within the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…We studied the ability of the ENV TMD to target the TLR2 TMD as it naturally resides in the membrane, and the TMDs of TLR2/6 were shown to be important for their regulation and function. The GALLEX system [23,24] was utilized to evaluate the hetero-association of gp41 and TLR2 TMDs in biological membranes. b-gal activity was measured after the expression of LexA-TMD-MBP chimera proteins in which the TMDs are those of ENV (wild type and mutant) and of TLR2.…”

Section: The Tmds Of Gp41 and Tlr2 Interact Within The Membrane Invomentioning

confidence: 99%

The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses

et al. 2014

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HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

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“…First, we show that the CA XII TM domain does indeed have an intrinsic interaction propensity in vivo. Interaction of the wt and mutated CA XII TM segment was analyzed using the GALLEX-system, a bacterial two-hybrid system, allowing the analysis of TM helix–helix interactions within a biological membrane [ 31 , 32 ]. While the CA XII TM domain forms a strong TM helix dimer in a biological membrane, single or multiple mutations of small residue(s) to the β-branched amino acid Ile almost always led to increased interaction propensities.…”

Section: Introductionmentioning

confidence: 99%

Small Residues Inhibit Homo-Dimerization of the Human Carbonic Anhydrase XII Transmembrane Domain

Cymer

Schneider

2021

Membranes

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Amino acids with small side chains and motifs of small residues in a distance of four are rather abundant in human single-span transmembrane helices. While interaction of such helices appears to be common, the role of the small residues in mediating and/or stabilizing transmembrane helix oligomers remains mostly elusive. Yet, the mere existence of (small)xxx(small) motifs in transmembrane helices is frequently used to model dimeric TM helix structures. The single transmembrane helix of the human carbonic anhydrases XII contains a large number of amino acids with small side chains, and critical involvement of these small amino acids in dimerization of the transmembrane domain has been suggested. Using the GALLEX assay, we show here that the transmembrane domain indeed forms a strong transmembrane helix oligomer within a biological membrane. However, single or multiple mutations of small residue(s) to isoleucine almost always increased, rather than decreased, the interaction propensities. Reduction of helix flexibility and of protein–lipid contacts caused by a reduced lipid accessible surface area likely results in stabilization of helix–helix interactions within the membrane.

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Analyzing Oligomerization of Individual Transmembrane Helices and of Entire Membrane Proteins in E. coli: A Hitchhiker’s Guide to GALLEX

Cited by 9 publications

References 28 publications

Six amino acids define a minimal dimerization sequence and stabilize a transmembrane helix dimer by close packing and hydrogen bonding

Six amino acids define a minimal dimerization sequence and stabilize a transmembrane helix dimer by close packing and hydrogen bonding

The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses

Small Residues Inhibit Homo-Dimerization of the Human Carbonic Anhydrase XII Transmembrane Domain

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