Article

Locating ligand binding and activation of a single antiporter.

BIOTEC, University of Technology, Tatzberg 47, 01062 Dresden, Germany.
EMBO Reports (Impact Factor: 7.19). 08/2005; 6(7):668-74. DOI: 10.1038/sj.embor.7400455
Source: PubMed

ABSTRACT Single-molecule force spectroscopy was applied to unfold individual Na(+)/H(+) antiporters NhaA from membrane patches. The force-extension curves contained detailed information about the strength and location of molecular interactions established within NhaA. Although molecular interactions that stabilize secondary structure elements remained unaffected on switching NhaA into its functional state, those that are assigned to the Na(+)-binding site changed markedly. These interactions were formed only in the presence of Na(+), with their full strength being established at pH approximately 6. This finding is in apparent contrast to measurements that suggest that NhaA is fully active at pH 7. Statistical analysis, however, showed that not all NhaA molecules activated this molecular interaction at pH 6, but at pH 7. This implies that the molecular interactions established on Na(+) binding may represent an early step in NhaA activation. The direct observation of molecular interactions established within an antiporter provides new insights into their activation mechanisms.

1 Bookmark
 · 
75 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H(+) across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H(+)-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H(+)- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H(+) symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.
    Proceedings of the National Academy of Sciences 04/2014; · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Structural topology plays an important role in protein mechanical stability. Proteins with β-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and 10FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with β-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a "mechanical clamp". Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to β-sandwich topology consisting of Greek key motifs but its overall structure lacks the "mechanical clamp" geometry at the termini. M-crystallin is a Ca2+ binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens β and γ-crystallins. We constructed an octamer of crystallin, (M-crystallin)8, and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ∼90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the β-sandwich topology proteins with a different strand-connectivity than that of I27 and 10FNIII, as well as lacking "mechanical clamp" geometry, can be mechanically resistant. Furthermore, Ca2+ binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ∼35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca2+ binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape.
    PLoS ONE 01/2014; 9(4):e94513. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In this paper we present a comparative study of supported lipid bilayers (SLBs) and proteolipid sheets (PLSs) obtained from deposition of lactose permease (LacY) of Escherichia coli proteoliposomes in plane. Lipid matrices of two components, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), at a 3:1, mol/mol ratio, were selected to mimic the inner membrane of the bacteria. The aim was to investigate how species of different compactness and stiffness affect the integration, distribution and nanomechanical properties of LacY in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or 1,2-palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG). Both compositions displayed phase separation and were investigated by atomic force microscopy (AFM) imaging and force-spectroscopy (FS) mode. PLSs displayed two separated, segregated domains with different features that were characterised by FS and force-volume mode. We correlated the nanomechanical characteristics of solid-like gel phase (Lβ) and fluid liquid-crystalline phase (Lα) with phases emerging in presence of LacY. We observed that for both compositions, the extended PLSs showed a Lβ apparently formed only by lipids, whilst the second domain was enriched in LacY. The influence of the lipid environment on LacY organisation was studied by performing protein unfolding experiments using the AFM tip. Although the pulling experiments were unspecific, positive events were obtained, indicating the influence of the lipid environment when pulling the protein. A possible influence of the lateral surface pressure on this behaviour is suggested by the higher force required to pull LacY from DPPE:POPG than from POPE:POPG matrices. This is related to higher forces governing protein-lipid interaction in presence of DPPE.
    Biochimica et Biophysica Acta 12/2013; · 4.66 Impact Factor

Full-text

Download
46 Downloads
Available from
Jun 10, 2014