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The COVID-19 pandemic emphasized the importance of peer communication networks for student outcomes. Herein, we describe the use of supplemental instructional videos created by former students and integrated into electronic lab notebooks, to restore the lost community-learning component using student voices in a de-densified upper-division biochemistry laboratory.

Increasing resistance against available orthosteric beta-lactamase inhibitors necessitates the search for novel and powerful inhibitor molecules. In this respect, allosteric inhibitors serve as attractive alternatives. Here, we examine the structural basis of inhibition in a hidden, druggable pocket in TEM-1 betalactamase. Based on crystallographic evidence that 6-cyclohexyl-1-hexyl-beta-D-maltoside (CYMAL-6) binds to this site, first we determined the kinetic mechanism of inhibition by CYMAL-6. Activity measurements with CYMAL-6 showed that it competitively inhibits the wild type enzyme. Interestingly, it exhibits a steep dose -response curve with an IC50 of 100 mu M. The IC50 value changes neither with different enzyme concentration nor with incubation of the enzyme with the inhibitor, showing that inhibition is not aggregation -based. The presence of the same concentrations of CYMAL-6 does not influence the activity of lactate dehydrogenase, further confirming the specificity of CYMAL-6 for TEM-1 beta-lactamase. Then, we identified compounds with high affinity to this allosteric site by virtual screening using Glide and Schrodinger Suite. Virtual screening performed with 500,000 drug like compounds from the ZINC database showed that top scoring compounds interact with the hydrophobic pocket that forms between H10 and Hll helices and with the catalytically important Arg244 residue through pi -cation interactions. Discovery of novel chemical scaffolds that target this allosteric site will pave the way for a new avenue in the design of new antimicrobials. (C) 2018 Elsevier Inc. All rights reserved.

The laboratory setting is an exciting and gratifying place to teach because you can actively engage the students in the learning process through hands-on activities; it is a dynamic environment amenable to collaborative work, critical thinking, problem-solving and discovery. The guided inquiry-based approach described here guides the students through their laboratory work at a steady pace that encourages them to focus on quality observations, careful data collection and thought processes surrounding the chemistry involved. It motivates students to work in a collaborative manner with frequent opportunities for feedback, reflection, and modification of their ideas. Each laboratory activity has four stages to keep the students' efforts on track: pre-lab work, an in-lab discussion, in-lab work, and a post-lab assignment. Students are guided at each stage by an instructor created template that directs their learning while giving them the opportunity and flexibility to explore new information, ideas, and questions. These templates are easily transferred into an electronic journal (termed the E-notebook) and form the basic structural framework of the final lab reports the students submit electronically, via a learning management system. The guided-inquiry based approach presented here uses a single laboratory activity for undergraduate Introductory Biochemistry as an example. After implementation of this guided learning approach student surveys reported a higher level of course satisfaction and there was a statistically significant improvement in the quality of the student work. Therefore we firmly believe the described format to be highly effective in promoting student learning and engagement. © 2013 by The International Union of Biochemistry and Molecular Biology, 42(1):58–67, 2014.

Many in vitro experiments aimed at studying the response of thiol-containing proteins to changes in environmental redox potentials use dithiothreitol (DTT) to maintain a preset redox environment throughout the experiments. However, the gradual oxidation of DTT during the course of the experiments, as well as the interaction between DTT and other components in the system, can significantly alter the initial redox potential and complicate data interpretation. Having an internal reporter for the actual redox potential of the assayed sample, facilitates the direct correlation between biochemical findings and the experimental redox status. Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) is a widely used, well-established tool for the analysis and purification of biomolecules, including proteins and peptides. Here, we describe a simple, robust, and quantitative RP-HPLC method we developed and tested to determine the experimental redox potential of an in vitro sample at the time of the experiment. It exploits the specific UV-absorbance of the oxidized intrinsic DTT in the samples and retains the high resolving power and high sensitivity of RP-HPLC with UV detection.

Declining efficiency of antibiotic-inhibitor combinatorial therapies in treating beta-lactamase mediated resistance necessitates novel inhibitor development. Allosteric inhibition offers an alternative to conventional drugs that target the conserved active site. Here, we show that the evolutionarily conserved PWP triad located at the N-terminus of the H10 helix directly interacts with the allosteric site in TEM-1 beta-lactamase and regulates its activity. While point mutations in the PWP triad preserve the overall secondary structures around the allosteric site, they result in a more open and dynamic global structure with decreased chemical stability and increased aggregation propensity. These mutant enzymes with a less compact hydrophobic core around the allosteric site displayed significant activity loss. Detailed sequence and structure conservation analyses revealed that the PWP triad is an evolutionarily conserved motif unique to class A beta-lactamases aligning its allosteric site and hence is an effective potential target for enzyme regulation and selective drug design.

Villin-type headpiece domains are compact motifs that have been used extensively as model systems for protein folding. Although the majority of headpiece domains bind actin, there are some that lack this activity. Here, we present the first NMR solution structure and 15N-relaxation analysis of a villin-type headpiece domain natively devoid of F-actin binding activity, that of supervillin headpiece (SVHP). The structure was found to be similar to other headpiece domains that bind F-actin. Our NMR analysis demonstrates that supervillin headpiece lacks a conformationally flexible region (V-loop), present in all other villin-type headpiece domains, and which is essential to the phosphoryl regulation of dematin headpiece. In comparing the electrostatic surface potential map of SVHP, to that of other villin-type headpiece domains with significant affinity for F-actin, we identified a positive surface potential conserved among headpiece domains that bind F-actin, but absent from SVHP. A single point mutation (L38K) in SVHP, which creates a similar positive surface potential, endowed SVHP with specific affinity for F-actin that is within an order of magnitude of the tightest binding headpiece domains. We propose that this effect is likely conferred by a specific buried salt-bridge between headpiece and actin. As no high-resolution structural information exists for the villin-type headpiece: F-actin complex, our results demonstrate that through positive mutagenesis, it is possible to design binding activity into homologous proteins without structural information of the counterpart's binding surface.

Notch receptors transmit signals between adjacent cells. Signaling is initiated when ligand binding induces metalloprotease cleavage of Notch within an extracellular negative regulatory region (NRR). We present here the X-ray structure of the human NOTCH2 NRR, which adopts an autoinhibited conformation. Extensive interdomain interactions within the NRR bury the metalloprotease site, showing that a substantial conformational movement is necessary to expose this site during activation by ligand. Leukemia-associated mutations in NOTCH1 probably release autoinhibition by destabilizing the conserved hydrophobic core of the NRR.

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase–tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I–positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

The laboratory setting is an exciting and gratifying place to teach because you can actively engage the students in the learning process through hands-on activities; it is a dynamic environment amenable to collaborative work, critical thinking, problem-solving and discovery. The guided inquiry-based approach described here guides the students through their laboratory work at a steady pace that encourages them to focus on quality observations, careful data collection and thought processes surrounding the chemistry involved. It motivates students to work in a collaborative manner with frequent opportunities for feedback, reflection, and modification of their ideas. Each laboratory activity has four stages to keep the students' efforts on track: pre-lab work, an in-lab discussion, in-lab work, and a post-lab assignment. Students are guided at each stage by an instructor created template that directs their learning while giving them the opportunity and flexibility to explore new information, ideas, and questions. These templates are easily transferred into an electronic journal (termed the E-notebook) and form the basic structural framework of the final lab reports the students submit electronically, via a learning management system. The guided-inquiry based approach presented here uses a single laboratory activity for undergraduate Introductory Biochemistry as an example. After implementation of this guided learning approach student surveys reported a higher level of course satisfaction and there was a statistically significant improvement in the quality of the student work. Therefore we firmly believe the described format to be highly effective in promoting student learning and engagement.

Background Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. Principal Findings The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. Conclusions/Significance S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage.

Proteolytic resistance of Notch prior to ligand binding depends on the structural integrity of a negative regulatory region (NRR) of the receptor that immediately precedes the transmembrane segment. The NRR includes the three Lin12/Notch repeats and the juxtamembrane heterodimerization domain, the region of Notch1 most frequently mutated in T cell acute lymphocytic leukemia. Here, we report the X-ray structure of the Notch1 NRR determined to 2.0 Å resolution in its autoinhibited conformation. A key feature of the Notch1 structure that maintains its closed conformation is a conserved hydrophobic plug that sterically occludes the metalloprotease cleavage site. Crystal packing interactions involving a highly conserved, exposed face on the third Lin12/Notch repeat suggest that this site may normally be engaged in inter- or intra-molecular protein-protein interactions. The vast majority of known T-ALL-associated point mutations of human Notch1 map to residues in the hydrophobic interior of the NRR. A novel mutation (H1545P), which alters a residue at the crystal-packing interface, leads to ligand-independent increases in signaling in reporter gene assays despite only mild destabilization of the NRR, suggesting that it releases the autoinhibitory clamp on the heterodimerization domain imposed by the Lin12/Notch repeats. The Notch1 NRR structure should facilitate a search for antibodies or compounds that stabilize the autoinhibited conformation.

Notch proteins are transmembrane proteins involved in controlling cell differentiation, cell growth, and cell death. An extracellular negative regulatory region (NRR) contains three contiguous LIN-12/Notch Repeats (LNRs) - LNRA, LNRB, and LNRC. Previous work from our lab showed that the total number of residues N-terminal to the first cysteine residue from human Notch1 LNRB (hN1LNRB) was critical for correct disulfide bond formation. Because of its more central location in relation to the rest of the protein, hN1LNRB participates in extensive interactions via its hydrophobic residues in the context of the full Notch protein. When expressed and folded in isolation, some of these residues are expected to be exposed to solvent and possibly contribute to the requirement of the additional N-terminal residues for autonomous in vitro folding with formation of the correct disulfide bonds. To test this hypothesis we mutated W52 to A52 and compared its folding pattern to that of the wild-type hN1LNRB. The effect of the total number of disulfide bonds on the autonomous folding of hN1LNRB was also investigated. In this study, the first of three pairs of disulfide bonds in hN1LNRB was eliminated by mutating C45 and C69 to A. Two mutant forms with two pairs of disulfide bonds were folded in vitro under the same conditions as the wild-type and the folding patterns were compared. The data and comparative analysis we present in this work demonstrate the importance of specific hydrophobic interactions and the total number of disulfide bonds as key determinants for the correct folding of an LNR module in addition to the total number of amino acids and Ca2+ ion coordination within the module.

Notch receptors are multi-domain trans-membrane proteins that are important for cell-cell communication and development. Deregulated Notch signaling has been linked to many human diseases such as sclerosis, artereopathy and leukemia. The extra-cellular domain of the Notch Receptor contains the Ligand Binding Domain and the Negative Regulatory Region (NRR), which includes three Lin-12/Notch Repeats (LNR), small disulfide-rich sequences of 35 residues. It has been previously shown that the first LNR from human Notch1, hN1LNRA, requires Ca2+ and a certain reduction potential that ensures the correct formation of three specific disulfide bonds believed to be critical for LNR structure and function. However, the first LNR in human Notch 4 and some of the LNRs found in PAPP (pregnancy-associated plasma protein-A), only possess four cysteines thereby can only form two disulfide bonds.In this work we present our findings on the effect of various reduction potentials as well as the elimination of the first disulfide bond in the in vitro folding of hN1 LNRA through a comparative analysis. The kinetics of the folding process for both the wild-type and the four-cysteine mutant form of hN1LNRA is studied by trapping various folding intermediates in a time-course manner, which is possible due to the slow rate of disulfide bond formation. Our results indicate that even though the wild-type hN1LNRA is very tolerant to variations in the specific redox potential in obtaining its ultimate correct folding, the its folding kinetics is significantly impacted. This is in contrast to the mutant form, which does not fold into a single species under identical refolding conditions.

Nat. Struct. Mol. Biol. 14, 295–300 (2007); published online 1 April 2007; corrected after print 1 April 2007 In the version of this article initially published, three references were missing from the reference list. The corrected reference list is shown below, and references have been renumbered accordingly.

The integral role that Notch proteins play in the normal development and tissue homeostasis of metazoan animals is orchestrated through the tightly regulated proteolytic processing of its Negative Regulatory Region (NRR). The NRR is composed of three linear notch repeats (LNR) followed by a heterodimerization (HD) domain that is N-terminal to the transmembrane region. Proteolytic Notch processing begins during transport to the cell surface with the cleavage of the HD domain at site S1 by a furin-like protease so as to produce a non-covalent heterodimer of one extracellular (NEC) and one transmembrane (NTM) subunit. Ligand binding at NEC initiates Notch activation by facilitating ADAM-type-metalloprotease-dependent extracellular cleavage at an NTM site (S2) that enables a subsequent intramembrane γ-secretase-mediated cleavage at site S3. These cleavages permit the translocation of the intracellular domain of Notch from the cell membrane to the nucleus to activate transcription. In this work we present the algorithms we have developed to evaluate the relative importance of specific amino acids for the structural stability and functionality of the NRR. These include predictive models derived from bioinformatic analysis such as phylogenetic inference and computational protein modeling as well as experimental data from multiple biophysical and biochemical techniques such as differential scanning calorimetry (DSC), circular dichroism (CD), and pull-down assays. Our results provide valuable insight to the mechanism of action of Notch.