Evolution of a self-renewing, participant-centered workshop series in BMB assessment.

We present as a case study the evolution of a series of participant-centered workshops designed to meet a need in the life sciences education community-the incorporation of best practices in the assessment of student learning. Initially, the ICABL (Inclusive Community for the Assessment of Biochemistry and Molecular Biology/BMB Learning) project arose from a grass-roots effort to develop material for a national exam in biochemistry and molecular biology. ICABL has since evolved into a community of practice in which participants themselves-through extensive peer review and reflection-become integral stakeholders in the workshops. To examine this evolution, this case study begins with a pilot workshop supported by seed funding and thoughtful programmatic assessment, the results of which informed evidence-based changes that, in turn, led to an improved experience for the community. Using participant response data, the case study also reveals critical features for successful workshops, including participant-centered activities and the value of frequent peer review of participants' products. Furthermore, we outline a train-the-trainer model for creating a self-renewing community by bringing new perspectives and voices into an existing core leadership team. This case study, then, offers a blueprint for building a thriving, evolving community of practice that not only serves the needs of individual scientist-educators as they seek to enhance student learning, but also provides a pathway for elevating members to positions of leadership.

Development of a Certification Exam to Assess Undergraduate Students' Proficiency in Biochemistry and Molecular Biology Core Concepts.

With support from the American Society for Biochemistry and Molecular Biology (ASBMB), a community of biochemistry and molecular biology (BMB) scientist-educators has developed and administered an assessment instrument designed to evaluate student competence across four core concept and skill areas fundamental to BMB. The four areas encompass energy and metabolism; information storage and transfer; macromolecular structure, function, and assembly; and skills including analytical and quantitative reasoning. First offered in 2014, the exam has now been administered to nearly 4000 students in ASBMB-accredited programs at more than 70 colleges and universities. Here, we describe the development and continued maturation of the exam program, including the organic role of faculty volunteers as drivers and stewards of all facets: content and format selection, question development, and scoring.

Assessing stakeholder perceptions of the american society for biochemistry and molecular biology accreditation program for baccalaureate degrees.

The American Society for Biochemistry and Molecular Biology (ASBMB) began an accreditation program in 2013. The criteria for accreditation of undergraduate programs include sufficient infrastructure - number and expertise of faculty, physical space and equipment, support for faculty and students - and incorporation of core concepts in the curriculum - structure and function of biomolecules; information storage; energy transfer; and quantitative skills. Students in accredited programs are able to have their degrees ASBMB certified by taking an exam focused on knowledge or skills across the four core concept areas. Members of the accreditation committees administered a survey to key stakeholders in the BMB community: undergraduate programs, both those that have applied for accreditation and those that have not; alumni/ae of accredited programs; graduate and professional programs; and employers. The goals of the study were to gauge the success of the program and determine necessary areas of improvement. The results indicate that the major benefits of applying for accreditation are the impetus to gather data and analysis not generally collected, and access to assessment data via the exam. However, stakeholders outside of the undergraduate community showed little awareness of the accreditation program. Additionally, the application process itself was seen to be very time consuming. This feedback will be used to improve the process and engage in further outreach. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):464-471, 2018.

Activation of SsoPK4, an Archaeal eIF2α Kinase Homolog, by Oxidized CoA.

The eukaryotic protein kinase (ePK) paradigm provides integral components for signal transduction cascades throughout nature. However, while so-called typical ePKs permeate the Eucarya and Bacteria, atypical ePKs dominate the kinomes of the Archaea. Intriguingly, the catalytic domains of the handful of deduced typical ePKs from the archaeon Sulfolobus solfataricus P2 exhibit significant resemblance to the protein kinases that phosphorylate translation initiation factor 2α (eIF2α) in response to cellular stresses. We cloned and expressed one of these archaeal eIF2α protein kinases, SsoPK4. SsoPK4 exhibited protein-serine/threonine kinase activity toward several proteins, including the S. solfataricus homolog of eIF2α, aIF2α. The activity of SsoPK4 was inhibited in vitro by 3',5'-cyclic AMP (Ki of ~23 µM) and was activated by oxidized Coenzyme A, an indicator of oxidative stress in the Archaea. Activation enhanced the apparent affinity for protein substrates, Km, but had little effect on Vmax. Autophosphorylation activated SsoPK4 and rendered it insensitive to oxidized Coenzyme A.

Protein Ser/Thr/Tyr phosphorylation in the Archaea.

The third domain of life, the Archaea (formerly Archaebacteria), is populated by a physiologically diverse set of microorganisms, many of which reside at the ecological extremes of our global environment. Although ostensibly prokaryotic in morphology, the Archaea share much closer evolutionary ties with the Eukarya than with the superficially more similar Bacteria. Initial genomic, proteomic, and biochemical analyses have revealed the presence of "eukaryotic" protein kinases and phosphatases and an intriguing set of serine-, threonine-, and tyrosine-phosphorylated proteins in the Archaea that may offer new insights into this important regulatory mechanism.

The activity of an ancient atypical protein kinase is stimulated by ADP-ribose in vitro.

The piD261/Bud32 protein kinases are universal amongst the members of the Eucarya and Archaea. Despite the fact that phylogenetic analyses indicate that the piD261/Bud32 protein kinases descend directly from the primordial ancestor of the "eukaryotic" protein kinase superfamily, our knowledge of their physiological role is relatively fragmentary and largely limited to two eucaryal representatives: piD261/Bud32 from yeast and the p53-related protein kinase from humans. A deduced archaeal homolog, SsoPK5, is encoded by open reading frame sso0433 from the acidothermophile Sulfolobus solfataricus. Recombinantly-expressed SsoPK5 exhibited protein kinase activity, with a noticeable preference for phosphorylating proteins of acidic character and for Mn(2+) as cofactor. The protein kinase also can phosphorylate itself on serine and threonine residues. The activity of rSsoPK5 was increased several-fold upon preincubation with either millimolar concentrations of 5'-AMP or submicromolar concentrations of ADP-ribose. Other mono- and di-nucleotides were ineffective. While activation was enhanced by the presence of ATP, no autophosphorylation of the protein kinase could be detected prior to addition of exogenous substrate proteins. We therefore suggest that ADP-ribose acts by evoking a conformational transition in the enzyme. Activation by ADP-ribose represents a potential regulatory link between chromatin remodeling and the activity of SsoPK5.

A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates.

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylated exogenous protein substrates. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Kinetic analysis indicated that the K(m) and V(max) values for SynPTP towards p-nitrophenyl phosphate are similar to those of other known bacterial low molecular weight PTPs. Mutagenic alteration of the predicted catalytic cysteine of PTP, Cys(7), to serine abolished enzyme activity. Using a combination of immunodetection, mass spectrometric analysis and mutagenically altered Cys(7)SerAsp(125)Ala-SynPTP, we identified PsaD (photosystem I subunit II), CpcD (phycocyanin rod linker protein) and phycocyanin-α and -ß subunits as possible endogenous substrates of SynPTP in this cyanobacterium. These results indicate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803.

A PPM-family protein phosphatase from the thermoacidophile Thermoplasma volcanium hydrolyzes protein-bound phosphotyrosine.

The genomes of virtually all free-living archaeons encode one or more deduced protein-serine/threonine/tyrosine kinases belonging to the so-called eukaryotic protein kinase superfamily. However, the distribution of their cognate protein-serine/threonine/phosphatases displays a mosaic pattern. Thermoplasma volcanium is unique among the Archaea inasmuch as it is the sole archaeon whose complement of deduced phosphoprotein phosphatases includes a member of the PPM-family of protein-serine/threonine phosphatases--a family that originated in the Eucarya. A recombinant version of this protein, TvnPPM, exhibited protein-tyrosine phosphatase in addition to its predicted protein-serine/threonine phosphatase activity in vitro. TvnPPM is the fourth member of the PPM-family shown to exhibit such dual-specific capability, suggesting that the ancestral versions of this enzyme exhibited broad substrate specificity. Unlike most other archaeons, the genome of T. volcanium lacks open reading frames encoding stereotypical protein-tyrosine phosphatases. Hence, the dual-specificity of TvnPPM may account for its seemingly aberrant presence in an archaeon.

The protein phosphatases of Synechocystis sp. strain PCC 6803: open reading frames sll1033 and sll1387 encode enzymes that exhibit both protein-serine and protein-tyrosine phosphatase activity in vitro.

The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp608 by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product of ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg2+ or Mn2+, for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.

A phosphohexomutase from the archaeon Sulfolobus solfataricus is covalently modified by phosphorylation on serine.

A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli. The recombinant protein proved capable of interconverting mannose 1-phosphate and mannose 6-phosphate, as well as glucose 1-phosphate and glucose 6-phosphate, in vitro. It displayed no catalytic activity toward glucosamine 6-phosphate or N-acetylglucosamine 6-phosphate. Models constructed using the X-ray crystal structure of a homologous phosphohexomutase from Pseudomonas aeruginosa predicted that Ser-309 of the archaeal protein lies within the substrate binding site. The presence of a phosphoryl group at this location would be expected to electrostatically interfere with the binding of negatively charged phosphohexose substrates, thus attenuating the catalytic efficiency of the enzyme. Using site-directed mutagenesis, Ser-309 was substituted by aspartic acid to mimic the presence of a phosphoryl group. The V(max) of the mutationally altered protein was only 4% that of the unmodified form. Substitution of Ser-309 with larger, but uncharged, amino acids, including threonine, also decreased catalytic efficiency, but to a lesser extent--three- to fivefold. We therefore predict that phosphorylation of the enzyme in vivo serves to regulate its catalytic activity.

The Phosphorylation Site Database: A guide to the serine-, threonine-, and/or tyrosine-phosphorylated proteins in prokaryotic organisms.

The Phosphorylation Site Database [http://vigen.biochem.vt.edu/xpd/xpd.htm] provides ready access to information from the primary scientific literature concerning those proteins from prokaryotic organisms, i.e., the members of the domains Archaea and Bacteria, that have been reported to undergo covalent phosphorylation on the hydroxyl side chains of serine, threonine, and/or tyrosine residues. Where known, the sequence of the site(s) of phosphorylation and the functional consequences of phosphorylation also are included. Active links enable users to quickly access further information concerning the phosphoprotein of interest from PubMed, GenBank, SWISS-PROT, and PIR.

A phosphoprotein from the archaeon Sulfolobus solfataricus with protein-serine/threonine kinase activity.

Sulfolobus solfataricus contains a membrane-associated protein kinase activity that displays a strong preference for threonine as the phospho-acceptor amino acid residue. When a partially purified detergent extract of the membrane fraction from the archaeon S. solfataricus that had been enriched for this activity was incubated with [gamma-(32)P]ATP, radiolabeled phosphate was incorporated into roughly a dozen polypeptides, several of which contained phosphothreonine. One of the phosphothreonine-containing proteins was identified by mass peptide profiling as the product of open reading frame [ORF] sso0469. Inspection of the DNA-derived amino acid sequence of the predicted protein product of ORF sso0469 revealed the presence of sequence characteristics faintly reminiscent of the "eukaryotic" protein kinase superfamily. ORF sso0469 therefore was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein formed insoluble aggregates that could be dispersed using urea or detergents. The solubilized polypeptide phosphorylated several exogenous proteins in vitro, including casein, myelin basic protein, and bovine serum albumin. Mutagenic alteration of amino acids predicted to be essential for catalytic activity abolished or severely reduced catalytic activity. Phosphorylation of exogenous substrates took place on serine and, occasionally, threonine. This new archaeal protein kinase displayed no catalytic activity when GTP was substituted for ATP as the phospho-donor substrate, while Mn(2+) was the preferred cofactor.

An arsenate reductase from Synechocystis sp. strain PCC 6803 exhibits a novel combination of catalytic characteristics.

The deduced protein product of open reading frame slr0946 from Synechocystis sp. strain PCC 6803, SynArsC, contains the conserved sequence features of the enzyme superfamily that includes the low-molecular-weight protein-tyrosine phosphatases and the Staphylococcus aureus pI258 ArsC arsenate reductase. The recombinant protein product of slr0946, rSynArsC, exhibited vigorous arsenate reductase activity (V(max) = 3.1 micro mol/min. mg), as well as weak phosphatase activity toward p-nitrophenyl phosphate (V(max) = 0.08 micro mol/min. mg) indicative of its phosphohydrolytic ancestry. pI258 ArsC from S. aureus is the prototype of one of three distinct families of detoxifying arsenate reductases. The prototypes of the others are Acr2p from Saccharomyces cerevisiae and R773 ArsC from Escherichia coli. All three have converged upon catalytic mechanisms involving an arsenocysteine intermediate. While SynArsC is homologous to pI258 ArsC, its catalytic mechanism exhibited a unique combination of features. rSynArsC employed glutathione and glutaredoxin as the source of reducing equivalents, like Acr2p and R773 ArsC, rather than thioredoxin, as does the S. aureus enzyme. As postulated for Acr2p and R773 ArsC, rSynArsC formed a covalent complex with glutathione in an arsenate-dependent manner. rSynArsC contains three essential cysteine residues like pI258 ArsC, whereas the yeast and E. coli enzymes require only one cysteine for catalysis. As in the S. aureus enzyme, these "extra" cysteines apparently shuttle a disulfide bond to the enzyme's surface to render it accessible for reduction. SynArsC and pI258 ArsC thus appear to represent alternative branches in the evolution of their shared phosphohydrolytic ancestor into an agent of arsenic detoxification.

Open reading frame sso2387 from the archaeon Sulfolobus solfataricus encodes a polypeptide with protein-serine kinase activity.

The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the "eukaryotic" protein kinase superfamily. sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures >or=60 degrees C, whereas phosphorylation of exogenous proteins was detectable at 37 degrees C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser(548), with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.

Phosphoprotein with phosphoglycerate mutase activity from the archaeon Sulfolobus solfataricus.

When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [gamma-(32)P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of approximately 46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co(2+) or Mn(2+). The recombinant protein underwent autophosphorylation when incubated with either [gamma-(32)P]ATP or [gamma-(32)P]GTP. The site of phosphorylation was identified as Ser(59), which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the (32)P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of (32)P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.

Archaeal protein kinases and protein phosphatases: insights from genomics and biochemistry.

Protein phosphorylation/dephosphorylation has long been considered a recent addition to Nature's regulatory arsenal. Early studies indicated that this molecular regulatory mechanism existed only in higher eukaryotes, suggesting that protein phosphorylation/dephosphorylation had emerged to meet the particular signal-transduction requirements of multicellular organisms. Although it has since become apparent that simple eukaryotes and even bacteria are sites of protein phosphorylation/dephosphorylation, the perception widely persists that this molecular regulatory mechanism emerged late in evolution, i.e. after the divergence of the contemporary phylogenetic domains. Only highly developed cells, it was reasoned, could afford the high 'overhead' costs inherent in the acquisition of dedicated protein kinases and protein phosphatases. The advent of genome sequencing has provided an opportunity to exploit Nature's phylogenetic diversity as a vehicle for critically examining this hypothesis. In tracing the origins and evolution of protein phosphorylation/dephosphorylation, the members of the Archaea, the so-called 'third domain of life', will play a critical role. Whereas several studies have demonstrated that archaeal proteins are subject to modification by covalent phosphorylation, relatively little is known concerning the identities of the proteins affected, the impact on their functional properties, or the enzymes that catalyse these events. However, examination of several archaeal genomes has revealed the widespread presence of several ostensibly 'eukaryotic' and 'bacterial' protein kinase and protein phosphatase paradigms. Similar findings of 'phylogenetic trespass' in members of the Eucarya (eukaryotes) and the Bacteria suggest that this versatile molecular regulatory mechanism emerged at an unexpectedly early point in development of 'life as we know it'.

The membrane-associated protein-serine/threonine kinase from Sulfolobus solfataricus is a glycoprotein.

Treatment of a sodium dodecyl sulfate-polyacrylamide gel with periodic acid-Schiff (PAS) stain or blotting with Galanthus nivalis agglutinin revealed the presence of several glycosylated polypeptides in a partially purified detergent extract of the membrane fraction of Sulfolobus solfataricus. One of the glycoproteins comigrated with the membrane-associated protein-serine/threonine kinase from S. solfataricus, which had been radiolabeled by autophosphorylation with [(32)P]ATP in vitro. Treatment with a chemical deglycosylating agent, trifluoromethanesulfonic acid, abolished PAS staining and reduced the M(r) of the protein kinase from approximately 67,000 to approximately 62,000. Protein kinase activity also adhered to, and could be eluted from, agarose beads containing bound G. nivalis agglutinin. Glycosylation of the protein kinase implies that at least a portion of this integral membrane protein resides on the external surface of the cell membrane.

Protein kinases and protein phosphatases in prokaryotes: a genomic perspective.

For many years, the regulation of protein structure and function by phosphorylation and dephosphorylation was considered a relatively recent invention that arose independently in each phylogenetic domain. Over time, however, incidents of apparent domain trespass involving the presence of 'eukaryotic' protein kinases or protein phosphatases in prokaryotic organisms were reported with increasing frequency. Today, genomics has provided the means to examine the phylogenetic distribution of 'eukaryotic' protein kinases and protein phosphatases in a comprehensive and systematic manner. The results of these genome searches challenge previous conceptions concerning the origins and evolution of this versatile regulatory mechanism.