Higher-order Structure Properties Research Articles

Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins.

Polymers of septin protein complexes play cytoskeletal roles in eukaryotic cells. The specific subunit composition within complexes controls functions and higher-order structural properties. All septins have globular GTPase domains. The other eukaryotic cytoskeletal NTPases strictly require assistance from molecular chaperones of the cytosol, particularly the cage-like chaperonins, to fold into oligomerization-competent conformations. We previously identified cytosolic chaperones that bind septins and influence the oligomerization ability of septins carrying mutations linked to human disease, but it was unknown to what extent wild-type septins require chaperone assistance for their native folding. Here we use a combination of in vivo and in vitro approaches to demonstrate chaperone requirements for de novo folding and complex assembly by budding yeast septins. Individually purified septins adopted nonnative conformations and formed nonnative homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble posttranslationally into native complexes. One septin, Cdc12, was so dependent on cotranslational chaperonin assistance that translation failed without it. Our findings point to distinct translation elongation rates for different septins as a possible mechanism to direct a stepwise, cotranslational assembly pathway in which general cytosolic chaperones act as key intermediaries.

Dynamics of the Tec-family tyrosine kinase SH3 domains.

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains.

Eye movements and variations in reading time.

Passages of prose were constructed which obeyed a stylistic convention referred to as ‘dovetailing’. For a given pair of sentences the final position of the first, and the initial position of the second, had the same referent. It was shown that critical dovetailed lines of text were read more rapidly than lines containing the same information, but lacking sequential structure. Degrading text by obscuring all interword spaces removed this reading time advantage. Analysis of subjects' eye movements during reading suggested that reading facilitation, induced by dovetailing, may result, not only from higher-order structural properties of text, but also from local features (particularly word-length information and associative relationships between words) known to influence eye movements.