Investigating evolutionary relationships through cluster analysis: A teaching science with big data workshop session.

Biochemistry is a data-heavy discipline, yet teaching students to work with large datasets is absent from many undergraduate Biochemistry programs. Ensuring that future generations of students arevbv confident in tackling problems using big data first requires that educators become comfortable teaching big data skills. The activity described herein introduces educators to working with big data and a framework for generating sequence similarity networks using JupyterLab and Python. This article reports a session from the virtual international 2021 IUBMB/ASBMB workshop, "Teaching Science with Big Data."

Modeling an Enzyme Active Site using Molecular Visualization Freeware.

Biomolecular visualization skills are paramount to understanding key concepts in the biological sciences, such as structure-function relationships and molecular interactions. Various programs allow a learner to manipulate 3D structures, and biomolecular modeling promotes active learning, builds computational skills, and bridges the gap between two dimensional textbook images and the three dimensions of life. A critical skill in this area is to model a protein active site, displaying parts of the macromolecule that can interact with a small molecule, or ligand, in a way that shows binding interactions. In this protocol, we describe this process using four freely available macromolecular modeling programs: iCn3D, Jmol/JSmol, PyMOL, and UCSF ChimeraX. This guide is intended for students seeking to learn the basics of a specific program, as well as instructors incorporating biomolecular modeling into their curriculum. The protocol enables the user to model an active site using a specific visualization program, or to sample several of the free programs available. The model chosen for this protocol is human glucokinase, an isoform of the enzyme hexokinase, which catalyzes the first step of glycolysis. The enzyme is bound to one of its substrates, as well as a non-reactive substrate analog, which allows the user to analyze interactions in the catalytic complex.

Meeting report: BioMolViz workshops for developing assessments of biomolecular visual literacy.

While molecular visualization has been recognized as a threshold concept in biology education, the explicit assessment of students' visual literacy skills is rare. To facilitate the evaluation of this fundamental ability, a series of NSF-IUSE-sponsored workshops brought together a community of faculty engaged in creating instruments to assess students' biomolecular visualization skills. These efforts expanded our earlier work in which we created a rubric describing overarching themes, learning goals, and learning objectives that address student progress toward biomolecular visual literacy. Here, the BioMolViz Steering Committee (BioMolViz.org) documents the results of those workshops and uses social network analysis to examine the growth of a community of practice. We also share many of the lessons we learned as our workshops evolved, as they may be instructive to other members of the scientific community as they organize workshops of their own.

SARS-CoV-2 virtual biochemistry labs on bioinformatics and drug design.

Colleges and universities are learning to provide relevant virtual lab experiences for students due to the COVID-19 pandemic. Even schools attempting in-person instruction often need to utilize virtual experiences for students absent due to quarantine or illness. Much of biochemistry is amenable to molecular visualization and/or computational study; however, many faculty face learning how to utilize new computational and molecular visualization software. We present a set of virtual lab exercises with detailed instructions to engage students in the discovery of novel antiviral compounds against the SARS-CoV-2 main protease.

De novo design of bioactive protein switches.

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.

Re-refinement of Plasmodium falciparum orotidine 5'-monophosphate decarboxylase provides a clearer picture of an important malarial drug target.

The development of antimalarial drugs remains a public health priority, and the orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) has great potential as a drug target. The crystallization of PfOMPDC with substrate bound represents an important advance for structure-based drug-design efforts [Tokuoka et al. (2008), J. Biochem. 143, 69-78]. The complex of the enzyme bound to the substrate OMP (PDB entry 2za1) would be of particular utility in this regard. However, re-refinement of this structure of the Michaelis complex shows that the bound ligand is the product rather than the substrate. Here, the re-refinement of a set of three structures, the apo enzyme and two versions of the product-bound form (PDB entries 2za1, 2za2 and 2za3), is reported. The improved geometry and fit of these structures to the observed electron density will enhance their utility in antimalarial drug design.

Proteolysis of truncated hemolysin A yields a stable dimerization interface.

Wild-type and variant forms of HpmA265 (truncated hemolysin A) from Proteus mirabilis reveal a right-handed, parallel ß-helix capped and flanked by segments of antiparallel ß-strands. The low-salt crystal structures form a dimeric structure via the implementation of on-edge main-chain hydrogen bonds donated by residues 243-263 of adjacent monomers. Surprisingly, in the high-salt structures of two variants, Y134A and Q125A-Y134A, a new dimeric interface is formed via main-chain hydrogen bonds donated by residues 203-215 of adjacent monomers, and a previously unobserved tetramer is formed. In addition, an eight-stranded antiparallel ß-sheet is formed from the flap regions of crystallographically related monomers in the high-salt structures. This new interface is possible owing to additional proteolysis of these variants after Tyr240. The interface formed in the high-salt crystal forms of hemolysin A variants may mimic the on-edge ß-strand positioning used in template-assisted hemolytic activity.

Enhancing student retention of prerequisite knowledge through pre-class activities and in-class reinforcement.

To foster the connection between biochemistry and the supporting prerequisite concepts, a collection of activities that explicitly link general and organic chemistry concepts to biochemistry ideas was written and either assigned as pre-class work or as recitation activities. We assessed student learning gains after using these activities alone, or in combination with regularly-integrated clicker and discussion questions. Learning gains were determined from student performance on pre- and post-tests covering key prerequisite concepts, biochemistry course exams, and student self-evaluation. Long-term retention of the material was assessed using a comprehensive exam given to a subset of the students. Our results show that using the pre-class exercises in combination with integrative questions was effective at improving student performance in both the short and long term. Similar results were obtained at both a large research institution with large class enrollments and at a private liberal arts college with moderate enrollments. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):97-104, 2017.

An expanded framework for biomolecular visualization in the classroom: Learning goals and competencies.

A thorough understanding of the molecular biosciences requires the ability to visualize and manipulate molecules in order to interpret results or to generate hypotheses. While many instructors in biochemistry and molecular biology use visual representations, few indicate that they explicitly teach visual literacy. One reason is the need for a list of core content and competencies to guide a more deliberate instruction in visual literacy. We offer here the second stage in the development of one such resource for biomolecular three-dimensional visual literacy. We present this work with the goal of building a community for online resource development and use. In the first stage, overarching themes were identified and submitted to the biosciences community for comment: atomic geometry; alternate renderings; construction/annotation; het group recognition; molecular dynamics; molecular interactions; monomer recognition; symmetry/asymmetry recognition; structure-function relationships; structural model skepticism; and topology and connectivity. Herein, the overarching themes have been expanded to include a 12th theme (macromolecular assemblies), 27 learning goals, and more than 200 corresponding objectives, many of which cut across multiple overarching themes. The learning goals and objectives offered here provide educators with a framework on which to map the use of molecular visualization in their classrooms. In addition, the framework may also be used by biochemistry and molecular biology educators to identify gaps in coverage and drive the creation of new activities to improve visual literacy. This work represents the first attempt, to our knowledge, to catalog a comprehensive list of explicit learning goals and objectives in visual literacy. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):69-75, 2017.

Sequential unfolding of the hemolysin two-partner secretion domain from Proteus mirabilis.

Protein secretion is a major contributor to Gram-negative bacterial virulence. Type Vb or two-partner secretion (TPS) pathways utilize a membrane bound ß-barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C-proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N-proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site-specific mutants of HpmA265. By correlating the effect of individual site-specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C-terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C-terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N-proximal polar core subdomain.

Cloning, expression, purification and characterization of an iron-dependent regulator protein from Thermobifida fusca.

Iron-dependent regulators (IdeRs) control the transcription of a variety of genes associated with iron homeostasis in Gram-positive bacteria. In this study we report the cloning of a putative IdeR gene from the moderate thermophile Thermobifida fusca into the pET-21a(+) expression vector. The expressed protein, Tf-IdeR, was purified using immobilized metal affinity and size-exclusion chromatography, and yielded approximately 12-16 mg of protein per liter of culture. The purified Tf-IdeR protein binds the tox operator sequence in the presence of divalent metal ions. Two Tf-IdeR binding sites were identified in the T. fusca genome upstream of a putative enterobactin exporter and a putative ABC-type multidrug transporter.

A bulky hydrophobic residue is not required to maintain the V-conformation of enzyme-bound thiamin diphosphate.

It is widely accepted that, in thiamin diphosphate (ThDP)-dependent enzymes, much of the rate acceleration is provided by the cofactor. Inter alia, the reactive conformation of ThDP, known as the V-conformation, has been attributed to the presence of a bulky hydrophobic residue located directly below the cofactor. Here we report the use of site-saturation mutagenesis to generate variants of this residue (Leu403) in benzoylformate decarboxylase. The observed 3 orders of magnitude range in k(cat)/K(m) values suggested that conformational changes in the cofactor could be influencing catalysis. However, X-ray structures of several variants were determined, and there was remarkably little change in ThDP conformation. Rather, it seemed that, once the V-conformation was attained, residue size and hydrophobicity were more important for enzyme activity.

Time-of-flight neutron diffraction study of bovine γ-chymotrypsin at the Protein Crystallography Station.

The overarching goal of this research project is to determine, for a subset of proteins, exact hydrogen positions using neutron diffraction, thereby improving H-atom placement in proteins so that they may be better used in various computational methods that are critically dependent upon said placement. In order to be considered applicable for neutron diffraction studies, the protein of choice must be amenable to ultrahigh-resolution X-ray crystallography, be able to form large crystals (1 mm(3) or greater) and have a modestly sized unit cell (no dimension longer than 100 Å). As such, γ-chymotrypsin is a perfect candidate for neutron diffraction. To understand and probe the role of specific active-site residues and hydrogen-bonding patterns in γ-chymotrypsin, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection. Time-of-flight neutron diffraction data were collected to 2.0 Å resolution at the PCS with ~85% completeness. Here, the first time-of-flight neutron data collection from γ-chymotrypsin is reported.

Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida.

Active sites may be regarded as layers of residues, whereby the residues that interact directly with substrate also interact with residues in a second shell and these in turn interact with residues in a third shell. These residues in the second and third layers may have distinct roles in maintaining the essential chemical properties of the first-shell catalytic residues, particularly their spatial arrangement relative to the substrate binding pocket, and their electrostatic and dynamic properties. The extent to which these remote residues participate in catalysis and precisely how they affect first-shell residues remains unexplored. To improve our understanding of the roles of second- and third-shell residues in catalysis, we used THEMATICS to identify residues in the second and third shells of the Co-type nitrile hydratase from Pseudomonas putida (ppNHase) that may be important for catalysis. Five of these predicted residues, and three additional, conserved residues that were not predicted, have been conservatively mutated, and their effects have been studied both kinetically and structurally. The eight residues have no direct contact with the active site metal ion or bound substrate. These results demonstrate that three of the predicted second-shell residues (α-Asp164, ß-Glu56, and ß-His147) and one predicted third-shell residue (ß-His71) have significant effects on the catalytic efficiency of the enzyme. One of the predicted residues (α-Glu168) and the three residues not predicted (α-Arg170, α-Tyr171, and ß-Tyr215) do not have any significant effects on the catalytic efficiency of the enzyme.

A preliminary neutron diffraction study of gamma-chymotrypsin.

The crystal preparation and preliminary neutron diffraction analysis of gamma-chymotrypsin are presented. Large hydrogenated crystals of gamma-chymotrypsin were exchanged into deuterated buffer via vapor diffusion in a capillary and neutron Laue diffraction data were collected from the resulting crystal to 2.0 A resolution on the LADI-III diffractometer at the Institut Laue-Langevin (ILL) at room temperature. The neutron structure of a well studied protein such as gamma-chymotrypsin, which is also amenable to ultrahigh-resolution X-ray crystallography, represents the first step in developing a model system for the study of H atoms in protein crystals.

D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily.

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.

Divergence of function in the thioredoxin fold suprafamily: evidence for evolution of peroxiredoxins from a thioredoxin-like ancestor.

The thioredoxin fold is found in proteins that serve a wide variety of functions. Among these are peroxiredoxins, which catalyze the reduction of hydrogen peroxide and alkyl peroxides. Although the common structural fold shared by thioredoxins and peroxiredoxins suggests the possibility that they have evolved from a common progenitor, it has been difficult to examine this hypothesis in depth because pairwise sequence identities between proteins in these two superfamilies are statistically insignificant. Using the Shotgun program, we have found that sequences of reductases involved in maturation of cytochromes in certain bacteria bridge the sequences of thioredoxins and peroxiredoxins. Analysis of motifs found in a divergent set of thioredoxins, cytochrome maturation proteins, and peroxiredoxins provides further support for an evolutionary relationship between these proteins. Within the conserved motifs are specific residues that are characteristic of individual protein classes, and therefore are likely to be involved in the specific functions of those classes. We have used this information, in combination with existing structural and functional information, to gain new insight into the structure-function relationships in these proteins and to construct a model for the emergence of peroxiredoxins from a thioredoxin-like ancestor.

Isoleucine 69 and valine 325 form a specificity pocket in human muscle creatine kinase.

Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by ATP. From a structural perspective, the enzyme utilizes two flexible loop regions to sequester and position the substrates for catalysis. There has been debate over the specific roles of the flexible loops in substrate specificity and catalysis in CK and other related phosphagen kinases. In CK, two hydrophobic loop residues, I69 and V325, make contacts with the N-methyl group of creatine. In this study, we report the alteration of the substrate specificity of CK through the mutagenesis of V325. The V325 to glutamate mutation results in a more than 100-fold preference for glycocyamine, while mutation of V325 to alanine results in a slight preference of the enzyme for cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). This study enhances our understanding of how the active sites of phosphagen kinases have evolved to recognize their respective substrates and catalyze their reactions.

Intersect: identification and visualization of overlaps in database search results.

UNLABELLED: The determination of distant evolutionary relationships remains an important biological problem, and distant homologs often appear in statistically insignificant regions of sequence similarity searches. Intersect is a computer program designed to identify and visualize the overlaps between sets of sequences reported by multiple database searches. This capability extends the usefulness of database search results and aids researchers in identifying the individual sequences that best bridge sequence families and superfamilies. AVAILABILITY: The Intersect program is available from the Babbitt laboratory website at http://www.babbittlab.ucsf.edu/software/intersect