Although tertiary folding of whole protein domains is prohibited by the cramped dimensions of the ribosomal tunnel, dynamic tertiary interactions may permit folding of small elementary units within the tunnel. To probe this possibility, we used a β-hairpin as well as an α-helical hairpin from the cytosolic N-terminus of a voltage-gated potassium channel and determined a probability of folding for each at defined locations inside and outside the tunnel. Minimalist tertiary structures can form near the exit port of the tunnel, a region that provides an entropic window for initial exploration of local peptide conformations. Tertiary subdomains of the nascent peptide fold sequentially, but not independently, during translation. These studies offer an approach for diagnosing the molecular basis for folding defects that lead to protein malfunction and provide insight into the role of the ribosome during early potassium channel biogenesis.

Mechanical forces acting on the ribosome can alter the speed of protein synthesis, indicating that mechanochemistry can contribute to translation control of gene expression. The naturally occurring sources of these mechanical forces, the mechanism by which they are transmitted 10 nm to the ribosome's catalytic core, and how they influence peptide bond formation rates are largely unknown. Here, we identify a new source of mechanical force acting on the ribosome by using in situ experimental measurements of changes in nascent-chain extension in the exit tunnel in conjunction with all-atom and coarse-grained computer simulations. We demonstrate that when the number of residues composing a nascent chain increases, its unstructured segments outside the ribosome exit tunnel generate piconewtons of force that are fully transmitted to the ribosome's P-site. The route of force transmission is shown to be through the nascent polypetide's backbone, not through the wall of the ribosome's exit tunnel. Utilizing quantum mechanical calculations we find that a consequence of such a pulling force is to decrease the transition state free energy barrier to peptide bond formation, indicating that the elongation of a nascent chain can accelerate translation. Since nascent protein segments can start out as largely unfolded structural ensembles, these results suggest a pulling force is present during protein synthesis that can modulate translation speed. The mechanism of force transmission we have identified and its consequences for peptide bond formation should be relevant regardless of the source of the pulling force.

The T1 recognition domains of voltage-gated K(+) (Kv) channel subunits form tetramers and acquire tertiary structure while still attached to their individual ribosomes. Here we ask when and in which compartment secondary and tertiary structures are acquired. We answer this question using biogenic intermediates and recently developed folding and accessibility assays to evaluate the status of the nascent Kv peptide both inside and outside of the ribosome. A compact structure (likely helical) that corresponds to a region of helicity in the mature structure is already manifest in the nascent protein within the ribosomal tunnel. The T1 domain acquires tertiary structure only after emerging from the ribosomal exit tunnel and complete synthesis of the T1-S1 linker. These measurements of ion channel folding within the ribosomal tunnel and its exit port bear on basic principles of protein folding and pave the way for understanding the molecular basis of protein misfolding, a fundamental cause of channelopathies.

Patch clamp technique was used to record cyclic nucleotide-dependent current of the frog olfactory receptor cell plasma membrane. Data obtained indicate that the channels passing this current are permeable to Ca 2+ or Mg 2+ and moderately selective for monovalent cations according to the sequence Li +, Na + , K + > Rb + > Cs + and are effectively blocked by l-c/s-diltiazem and Y,4'-dichlorobenzamil. The conductance of single cyclic nucleotide-gated channels in solutions with low Ca t+ and Mg 2+ content is about 19 pS. The results demonstrate that cyclic nucleotide-activated channels of olfactory receptor cells are virtually identical to photoreceptor ones.

Voltage-gated K ؉ channels (Kv) are tetramers whose assembly is coordinated in part by a conserved T1 recognition domain. Although T1 achieves its quaternary structure in the ER, nothing is known about its acquisition of tertiary structure. We developed a new folding assay that relies on intramolecular cross-linking of pairs of cysteines engineered at the folded T1 monomer interface. Using this assay, we show directly that the T1 domain is largely folded while the Kv protein is still attached to membrane-bound ribosomes. The ER membrane facilitates both folding and oligomerization of Kv proteins. We show that folding and oligomerization assays can be used to study coupling between these two biogenic events and diagnose defects in assembly of Kv channels.K ϩ channels comprise a diverse and ubiquitous class of membrane proteins designed to facilitate the diffusion of K ϩ ions across the plasma membrane. Among this class of channels are voltage-gated K ϩ channels (Kv), 1 which open and close in response to changes of membrane potential. Kv channels are formed by four subunits that surround the central permeation pathway. The monomeric Kv subunits assemble as tetrameric species in the ER membrane (1-7) and subsequently traffic to the plasma membrane. Once assembled, they do not dissociate in the plasma membrane (8), but it is not known whether they are in equilibrium with monomers in the ER membrane. Kv tetramerization likely occurs in a series of steps (6, 7, 9) involving different domains of the Kv protein and perhaps different biogenic intermediates. For example, the cytoplasmic T1 domain, an N-terminal sequence that is highly conserved among Kv channels and is responsible for subfamily-specific co-assembly of subunits (5, 10 -12), can self-associate before the monomeric nascent Kv channel peptide detaches from the ribosome and exits from the protein-translocating channel in the ER (translocon) (9). This self-association occurs between folded T1 monomers (9, 13), giving rise to intersubunit interfaces that contain ϳ20 side chains that are involved in polar intersubunit interactions (14, 15). As is common for oligomeric proteins, the tertiary structure of individual subunits confers the proper interface for quaternary structure formation. Therefore, the aforementioned polar side chains are only in close proximity when the monomer is folded.In this study, we focus on two biogenic events: folding of the T1 domain in the monomer and tetramerization of T1 domains. These two events must occur over the time frame of biogenesis. Folding into the tertiary structure could occur as soon as the T1 domain exits the ribosome tunnel into the cytosol, and tetramerization of T1 domains may be complete before Kv channels exit the ER membrane. The ability of T1 to form tetramers (tetramerization competence) could also begin when the nascent peptide first emerges from the ribosome. The interplay between T1 folding and tetramerization has not been explored, largely because of the unavailability of a folding assay. Previously, we sugg...