Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

55-POS

Board 27

DYNAMICS AND EXPANSION OF THE FLEXIBLE CAVITY OF THE PERIPLASMIC CHAPERONE SKP Mayank Gupta 1 ; Andreas Hartmann 1 ; Neharika Chamachi 1,2 ; Michael Schlierf 1,3 ; 1 TU Dresden, B CUBE, Dresden, Germany 2 TU Dresden, DRESDEN concept Genome Center, Dresden, Germany 3 TU Dresden, Faculty of Physics, Dresden, Germany Gram-negative bacteria are protected by an outer membrane packed with β -barrel outer membrane proteins (OMPs). OMPs perform diverse functions ranging from nutrient transport, signal transduction, host interaction and drug resistance, hence their assembly must be tightly regulated. After translocation across the inner membrane, OMPs enter the aqueous periplasm. Due to their hydrophobicity, OMPs are at risk of aggregation. The two major chaperones, SurA and Skp, prevent aggregation by holding OMPs in an extended conformation and delivering them to the β -barrel assembly machinery (BAM) for insertion into the outer membrane. Since disruption of OMP biogenesis compromises bacterial viability, this pathway is an attractive target for antibiotic development. Skp is a homo-trimer that holds an unfolded OMP within a central cavity. However, this cavity has been difficult to study because Skp trimers dissociate into inactive monomers at the low concentrations required for single-molecule experiments. As a result, fundamental questions regarding cavity opening and conformational dynamics upon binding to OMPs of different sizes remain unanswered. To overcome this limitation, we genetically engineered a single-chain Skp by linking the three subunits with flexible linkers into a single polypeptide. The recombinant protein retained its chaperone activity, and exhibited a higher affinity for OmpX, consistent with elimination of the monomer-trimer equilibrium. By introducing cysteine residues at defined positions within the single-chain construct, we can site specifically attach fluorescent dyes for single-molecule FRET measurements. This enables us to monitor conformational dynamics of the Skp cavity upon binding to OMP substrates.

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