Didem Vardar

The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain

Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35‐residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic–aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D‐NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair‐wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic–aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.

Visible photoluminescence from low temperature deposited hydrogenated amorphous silicon nitride

Abstract Hydrogenated amorphous silicon nitride (a-SiNx:H) sample have been prepared by plasma enhanced chemical vapor deposition (PECVD) using a mixture of silane (SiH4), nitrogen and ammonia (NH3). Most films exhibit visible photoluminescence (PL) and some emit strong PL after annealing. While films grown without NH3 exhibit PL in the deep red, those grown with NH3 show PL in the green. The PL properties of these films with no oxygen (O) content are similar to those of silicon oxide (SiOx) films and porous Si. Using infrared and X-ray Photoelectron Spectroscopy, we suggest that PL from a-SiNx:H films originate from Si clusters which form during PECVD and crystallize upon annealing. We propose that the presence of O is not necessary for efficient PL.

The NMR Structure of Dematin Headpiece Reveals a Dynamic Loop That Is Conformationally Altered upon Phosphorylation at a Distal Site

Dematin (band 4.9) is found in the junctional complex of the spectrin cytoskeleton that supports the erythrocyte cell membrane. Dematin is a member of the larger class of cytoskeleton-associated proteins that contain a modular “headpiece” domain at their extreme C termini. The dematin headpiece domain provides the second F-actin-binding site required for in vitro F-actin bundling. The dematin headpiece is found in two forms in the cell, one of 68 residues (DHP) and one containing a 22-amino acid insert near its N terminus (DHP+22). In addition, dematin contains the only headpiece domain that is phosphorylated, in vivo. The 22-amino acid insert in DHP+22 appeared unstructured in NMR spectra; therefore, we have determined the three-dimensional structure of DHP by multidimensional NMR methods. Although the overall three-dimensional structure of DHP is similar to that of the villin headpiece, there are two novel characteristics revealed by this structure. First, unlike villin headpiece that contains a single buried salt bridge, DHP contains a buried charged cluster comprising residues Glu39, Arg66, Lys70, and the C-terminal carboxylate of Phe76. Second, 15N relaxation experiments indicate that the longer “variable loop” region near the N terminus of DHP (residues 20–29) is dynamic, undergoing significantly greater motions that the rest of the structure. Furthermore, NMR chemical shift changes indicate that the conformation of the dynamic variable loop is altered by phosphorylation of serine 74, which is far in the sequence from the variable loop region. Our results suggest that phosphorylation of the dematin headpiece acts as a conformational switch within this headpiece domain.