Elucidating the Role of a Calcium-Binding Loop in an x-Prolyl Aminodipeptidase from Lb. helveticus.

Prolyl aminodipeptidase (PepX) is an α/ß hydrolase that cleaves at penultimate N-terminal prolyl peptide bonds. The crystal structure of PepX from Lactobacillus helveticus exhibits a calcium-binding loop within the catalytic domain. The calcium-binding sequence of xDxDxDGxxD within this loop is highly conserved in PepX proteins among lactic acid bacteria, but its purpose remains unknown. Enzyme activity is not significantly affected in the presence of the metal chelator ethylenediaminetetraacetic acid (EDTA), nor in the presence of excess calcium ions. To eliminate calcium binding, D196A and D194A/D196A mutations were constructed within the conserved calcium-binding sequence motif. Enzyme activity and stability of the D196A mutant were comparable to the wild-type enzyme by colorimetric kinetic assays and protein thermal shift assays. However, the D194A/D196A mutant was inactive though it retained native-like structure and thermal stability, contradicting the EDTA and calcium titration results. This suggests calcium binding to PepX may be essential for activity.

Structural characterization of a prolyl aminodipeptidase (PepX) from Lactobacillus helveticus.

Prolyl aminodipeptidase (PepX) is an enzyme that hydrolyzes peptide bonds from the N-terminus of substrates when the penultimate amino-acid residue is a proline. Prolyl peptidases are of particular interest owing to their ability to hydrolyze food allergens that contain a high percentage of proline residues. PepX from Lactobacillus helveticus was cloned and expressed in Escherichia coli as an N-terminally His-tagged recombinant construct and was crystallized by hanging-drop vapor diffusion in a phosphate buffer using PEG 3350 as a precipitant. The structure was determined at 2.0 Šresolution by molecular replacement using the structure of PepX from Lactococcus lactis (PDB entry 1lns) as the starting model. Notable differences between the L. helveticus PepX structure and PDB entry 1lns include a cysteine instead of a phenylalanine at the substrate-binding site in the position which confers exopeptidase activity and the presence of a calcium ion coordinated by a calcium-binding motif with the consensus sequence DX(DN)XDG.

Assessment of learning gains in a flipped biochemistry classroom.

The flipped classroom has become an increasingly popular pedagogical approach to teaching and learning. In this study, learning gains were assessed in a flipped biochemistry course and compared to gains in a traditional lecture. Although measured learning gains were not significantly different between the two courses, student perception of learning gains did differ and indicates a higher level of satisfaction with the flipped lecture format.

Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments.

Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3'-kinase fold into an active native-like structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18 degrees C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.