No abstract

Protein folding is dependent on the formation and persistence of simple loops during the earliest events of the folding process. Ease of loop formation and persistence is believed to be dependent on the steric properties of the residues involved in loop formation. We have investigated this conformational factor in the denatured state of iso-1-cytchrome c using a five alanine insert in front of a unique histidine in the N-terminal region of the protein. The alanine residues have then been progressively substituted with sterically less-constrained glycine residues. Guanidine-HCl unfolding shows that all variants have a free energy of unfolding of approximately 2 kcal/mol. The low stability of these variants is well accounted for by stabilization of the denatured state by histidine-heme loop formation. The stability of the 22 residue histidine-heme loop has been measured in 3 M guanidine hydrochloride for all variants. Surprisingly, relative to alanine, glycine has only a very modest effect on equilibrium loop stability. Thus, the greater flexibility that glycine confers on the main-chain provides no advantage in terms of the persistence of simple loops early in folding. The underlying basis for the similar behavior of loops with polyalanine versus polyglycine inserts is discussed in terms of the current knowledge of the structure and loop formation kinetics of glycine versus alanine-rich peptides.

The vertebrate nuclear pore protein Nup153 contains a novel RNA binding domain. This 150-amino acid region was previously found to bind preferentially to a panel of mRNAs when compared with structured RNAs, such as tRNA, U snRNA, and double-stranded RNA. The ability to broadly recognize mRNA led to the conclusion that the Nup153 RNA binding domain confers a general affinity for single-stranded RNA. Here, we have probed Nup153 RNA recognition to decipher how this unique RNA binding domain discriminates between potential targets. We first mapped the binding determinant within an RNA fragment that associates relatively robustly with the Nup153 RNA binding domain. We next designed synthetic RNA oligonucleotides to systematically delineate the features within this minimal RNA fragment that are key to Nup153 RNA-binding domain binding and demonstrated that the binding preferences of Nup153 do not reflect general preferences of an mRNA/single-stranded RNA-binding protein. We further found that the association between Nup153 and a cellular mRNA can be attributed to an interaction with specific subregions of the RNA. These results indicate that Nup153 can discriminate between mRNA and other classes of RNA transcripts due in part to direct recognition of a loose sequence motif. This information adds a new dimension to the interfaces that can contribute to recognition in mRNA export cargo selection and fate.Nuclear pore complexes are macromolecular structures that bridge the inner and outer nuclear membranes to form a channel for nucleocytoplasmic traffic (1-3). Recent molecular characterization of pore complexes has revealed that they are comprised of only ϳ30 different proteins (4, 5), with multiples of eight copies of each protein forming the 8-fold symmetric structure characteristic of the nuclear pore complex. A small number of nucleoporins are restricted in localization to either the nuclear or cytoplasmic side of the pore, and their skewed distribution contributes to distinct features on each of these faces: the nuclear basket structure and the cytoplasmic filaments. The observation that repetitive arrangement of a relatively limited number of proteins creates the elaborate pore structure suggests that each nucleoporin carries out multiple tasks, from providing structural scaffolding to contacting diverse cargo-receptor complexes as they transit through the pore.The paradigm of multifunctional pore components is well illustrated by the nucleoporin Nup153. This pore protein plays roles in both import and export pathways (6 -11). Consistent with this, Nup153 interacts with various transport receptors (7,(12)(13)(14)(15)(16)(17)(18), as well as with specific cargo (6, 10). Nup153 has also been found to be essential for the localization of other pore components (19) and is specifically thought to anchor the basket constituent TPR (20). Somewhat paradoxically, Nup153 has been shown to be dynamically localized to the nuclear pore (21,22). This apparent contradiction, as well as the multiple roles implicated for Nup153, ma...

The competition between intramolecular histidine-heme loop formation and ligand-mediated oligomer formation in the denatured state is investigated for two yeast iso-1-cytochrome c variants, AcH26I52 and AcA25H26I52. Besides the native His 18 heme ligand, both variants contain a single His at position 26. The AcA25H26I52 variant has Pro 25 mutated to Ala. The concentration dependence of the apparent pKa for His 26-heme binding in 3 M gdnHCl indicates that the P25A mutation disfavors oligomerization mediated by intermolecular heme ligation by 10-fold. Single and double pH jump stopped-flow experiments with the AcH26I52 variant show that fast phases for His-heme bond formation and breakage are due to intramolecular loop formation and slow phases for His-heme bond formation and breakage are due to intermolecular aggregation. The presence of two closely-spaced slow phases in the kinetics of loop formation for both variants suggests that intermolecular His 26-heme ligation results in both dimers and higher order aggregates. The P25A mutation slows formation and speeds breakdown of an initial dimer, demonstrating a strong effect of local sequence on aggregation. Analysis of the kinetic data yields equilibrium constants for intramolecular loop formation and intermolecular dimerization at pH 7.1 and indicates that the rate constant for intermolecular aggregation is very fast at this pH (107 to 108 M−1s−1). In light of the very fast rates of aggregation in the denatured state, comparison of models involving reversible or irreversible oligomerization steps suggest that equilibrium control of the partitioning between folding and aggregation is advantageous for productive protein folding in vivo.