Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome.

During the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of the Escherichia coli ribosomal protein S6 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6).

The function of the ATP-binding cassette transporter MRP6 is unknown but mutations in its gene cause pseudoxanthoma elasticum. We have investigated the membrane topology of the N-terminal transmembrane domain TMD0 of MRP6 and the membrane integration and orientation propensities of its transmembrane segments (TMs) by glycosylation mapping. Results demonstrate that TMD0 has five TMs, an Nout-Cin topology and that the less hydrophobic TMs have strong preference for their orientation in the membrane that affects the neighboring TMs. Two disease-causing mutations changing the number of positive charges in the loops of TMD0 did not affect the membrane insertion efficiencies of the adjacent TMs.