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.

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 Å resolution 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.

The N-terminal SH2 domain from the p85␣ subunit of phosphatidylinositol 3Ј kinase is cleaved specifically into 9-and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like 1 H-15 N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies.Keywords: Fragment complementation; fragment reconstitution; protein-protein interactions; protein folding; nuclear magnetic resonance spectroscopy; limited proteolysis Supplemental material: See www.proteinscience.org.Certain single-domain proteins show native-like structure and function as noncovalent complexes of their constituent peptide fragments. This complementation of domain fragments has been used to study protein-protein interactions in folding/binding events

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 p85␣ subunit of phosphatidylinositol 3Ј-kinase fold into an active nativelike 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°C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.Keywords: Fragment complementation; SH2 domain; electrostatic interactions; protein stability Supplemental material: See www.proteinscience.org.Extensive site-directed mutagenesis studies of intact proteins have been used to reveal the fundamental interactions that stabilize protein structures. Buried hydrophobic interactions clearly play an essential role in stabilizing specific folds (Richards 1977;Kellis et al. 1989;Sandberg and Terwilliger 1989;Bowie et al. 1990;Shortle et al. 1990;Lim and Sauer 1991;Eriksson et al. 1992;Jackson et al. 1993;Cordes et al. 1996;Dahiyat and Mayo 1997;Xu et al. 1998;Chen and Stites 2001), whereas the role of surface interactions is less clear Tidor 1994, 1999;Meeker et al. 1996;Suckow et al. 1996;Schwehm et al. 1998;Sindelar et al. 1998;Cordes and Sauer 1999;Xiao and Honig 1999;Spector et al. 2000;Strop and Mayo 2000;Perl and Schmid 2001). Fragment complementation is another useful and sensitive phenomenon that can be used to analyze the interactions that stabilize protein structures. Certain single domain proteins retain the ability to form a folded non-covalent complex when cleaved into one or more fragments (for review, see de Prat-Gay 1996). These complexes Reprint requests to: Deborah S. Wuttke, Department of Chemistry and Biochemistry, UCB 215, ...

Large residual (15)N-(1)H dipolar couplings have been measured in a Src homology II domain aligned at Pf1 bacteriophage concentrations an order of magnitude lower than used for induction of a similar degree of alignment of nucleic acids and highly acidic proteins. An increase in (1) H and (15)N protein linewidths and a decrease in T(2) and T(1)ρ relaxation time constants implicates a binding interaction between the protein and phage as the mechanism of alignment. However, the associated increased linewidth does not preclude the accurate measurement of large dipolar couplings in the aligned protein. A good correlation is observed between measured dipolar couplings and predicted values based on the high resolution NMR structure of the SH2 domain. The observation of binding-induced protein alignment promises to broaden the scope of alignment techniques by extending their applicability to proteins that are able to interact weakly with the alignment medium.