Single-molecule force spectroscopy of polycystic kidney disease proteins.

Atomic force microscopy in its single-molecule force spectroscopy mode is a nanomanipulation technique that is extensively used for the study of the mechanical properties of proteins. It is particularly suited to examine their response to stretching (i.e., molecular elasticity and mechanical stability). Here, we describe protein engineering strategies and single-molecule AFM techniques for probing protein mechanics, with special emphasis on polycystic kidney disease (PKD) proteins. We also provide step-by-step protocols for preparing proteins and performing single-molecule force measurements.

Atomic force microscopy reveals the alternating subunit arrangement of the TRPP2-TRPV4 heterotetramer.

There is evidence that polycystin-2 (TRPP2) interacts with two other members of the transient receptor potential (TRP) family, TRPC1 and TRPV4. We have previously shown that TRPP2 forms a heteromeric complex with TRPC1, with a 2:2 stoichiometry and an alternating subunit arrangement. Here, we used coimmunoprecipitation to show that TRPP2 also interacts with TRPV4, but not with TRPA1 or TRPM8; hence, its promiscuity is limited. We then used atomic force microscopy to study the structure of the TRPV4 homomer and the interaction between TRPP2 and TRPV4. The molecular volume of V5-tagged TRPV4 isolated from singly-transfected tsA 201 cells indicated that it assembled as a homotetramer. The distribution of angles between pairs of anti-V5 antibodies bound to TRPV4 particles had a large peak close to 90 degrees and a smaller peak close to 180 degrees , again consistent with the assembly of TRPV4 as a homotetramer. In contrast, the angle distributions for decoration of the TRPP2-TRPV4 heteromer by either anti-Myc or anti-V5 antibodies had major peaks close to 180 degrees. This result indicates that TRPP2-TRPV4 assembles identically to TRPP2-TRPC1, suggesting a common subunit arrangement among heteromeric TRP channels.