3D structures of biomolecules are critical to understanding molecular mechanisms in core biological processes. Visualization of these dynamic processes can be challenging for students. However, this skill is critical for advancing their knowledge and reinforcing core concepts beyond the textbook. Thus, protein structure–function relationships serve as a learning outcome in many undergraduate biochemistry and molecular biology courses. The RCSB PDB database entries has grown to over 200K entries in past decade with an average increase of about 12K each year. This database will continue to grow as the methods of structural biology improve along with technological advances. It implies that a structural understanding of biomolecules is no longer a specialized theme, but an integral part of the curriculum. Furthermore, the ability to effectively evaluate and navigate structural data are skills needed in the modern academic and industrial workforce. During COVID-19, we expanded the protein-structure-function exploration (PSFE) initiatives at our primarily undergraduate HBCU. We focused on implementing PyMOL to advance interactive learning in virtual setting during pandemic hardship coupled with interactive engagement during extended lab and lecture sessions. PyMOL provided a way to dive into protein structures via the graphical user interface using basic functions as well as advanced functions that required text commands that could be combined into elaborate analysis scripts using procedural programming. The use molecular graphics software has been instrumental to infusing structural interpretation of molecular mechanisms that can lead to the formulation of new hypotheses. During the pilot phase of our PSFE initiative, we developed a plan to infuse our research involving modeling a helical sensory receptor, an oligomeric beta sheet transducer, and DNA-protein interaction as an introduction to PyMOL using a guided tutorial. The initial assessment was focused using semiquantitative queries that probed a student's understanding and appreciation of visualization and analysis of 3D structures. A subsequent approach focused on expansion of the preliminary activity towards similar structures in RCSB PDB database and utilizing a tutorial in addition to the use structural bioinformatics. Students then prepared a detailed structural analysis in form of a written report. The outcomes of the pilot PSFE were reported at the 2021 ASBMB "Teaching with Big Data" conference and ultimately published [1]. Our PSFE initiatives have enhanced the rigor in our independent research, biochemistry, and molecular biology courses and labs. We have used crucial biomolecules in the central dogma to stimulate effective and inclusive engagement via visualization and structural approaches using PyMOL and the RCSB PDB database. Our initial results have shown increased not only increased performance in class, but also deeper and more meaningful discourse in their products. As we continue to improve our tutorials and methods, our future aims will include the use of more advanced features in PyMOL and develop modules that will reinforce these skills as well as implementation of the basic modules in earlier courses. [1] Trivedi,V.D.(2022) "PSFE[ProteinStructure-FunctionExploration]Initiativein Undergraduate Biochemistry and Independent Research Courses" Biochem. & Mol. Biol. Edu., 50 [5], 473-475. Supported by BCU STEM Excellence scholarship program and previous support of NIH-NIGMS SCORE SC3GM113803 [ended in 2020].

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N6-ethenoadenine (ϵA) lesions or 3,N4-ethenocytosine (ϵC) lesions, it can only excise ϵA lesions. AAG binds very tightly to DNA containing ϵC lesions, forming an abortive protein-DNA complex; such binding not only shields ϵC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing ϵC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the ϵC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for ϵC versus ϵA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O2 of ϵC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.

Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (76Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg−1 SeMet) was provided. Both high-performance liquid chromatography (HPLC)–ICP-MS or gas chromatography (GC)–ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg−1 (n = 13, with a standard deviation of 9.7 mg kg−1) and 561.5 mg kg−1(n = 11, with a standard deviation of 44.3 mg kg−1) with median values of 337.6 and 575.0 mg kg−1 for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n = 9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg−1 Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides (dNTPs) in all organisms and thus play an essential role in DNA biosynthesis and repair. This function makes RNRs attractive targets for anticancer and antiparasitic chemotherapies since inhibition of these enzymes disrupts the dNTP balance within the cell, leading to cell death. Previous work has shown that thioredoxin (Trx)/thioredoxin reductase systems are responsible for the re-reduction of class I and class II RNRs through disulfide exchange reactions involving conserved cysteine residues. To study this re-reduction reaction, we have selected proteins from Lactobacillus leichmannii, an ideal model system due to the relative simplicity of its ribonucleoside triphosphate reductase (RTPR) and the presence of only one Trx in its genome. We have cloned Trx from Lactobacillus leichmannii and purified the protein to homogeneity. To investigate which of the cysteine residues at the C-terminus of RTPR are more reactive toward Trx, we have prepared RTPR mutant proteins C731S and C736S. Further, to investigate which of the cysteines in the conserved Trx ‘CGPC’ motif are more reactive toward RTPR, we have prepared C28S and C31S Trx mutant proteins. Preliminary results of the disulfide-mediated cross-linking of RTPR with Trx will be presented. (Supported by National Institutes of Health GM65337)

Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)-cytochrome b(5) reductase (cytb(5)r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255- and G291D, have been identified in the NADH-binding lobe of cytb(5)r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845-48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb(5)r mutations were also identified, including the R159- mutation, which causes loss of the entire NADH-binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH-binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb(5)r. In contrast to the E255- and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb(5)r deficient patients who are heterozygous for an NADH-binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH-binding lobe.

The conserved sequence motif "RxY(S)(T)xx(N)(S)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(S)(T)xx(N)(S)" Motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor: protein stoichiornetry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K-m(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K-s (H4NAD) and K-s(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (Edegrees', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were similar to30 mV more positive than that of wild-type cb5r (Edegrees' = -268 mV) while that of Y93H was similar to30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an arornatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.

Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.

Deficiency of NADH-cytochrome b5 reductase (cb5r) causes two clinically distinct phenotypes of recessive congenital methemoglobinemia (RCM). Type I patients often manifest cyanosis from birth, and in type II patients the cyanosis is accompanied by severe neurological impairment. The mechanisms responsible for the phenotypic differences between the two subgroups remain to be defined. The majority of patients harbor two different mutant alleles. To date 39 mutant variants of cb5r have been identified, 2 of which are common to both types of RCM. In order to characterize the individual cb5r variant proteins we have developed a novel heterologous expression system based on the structures of the rat and human proteins derived by X-ray crystallography. The system permits the investigation of the catalytic efficiencies, protein thermostability, FAD cofactor properties and substrate (NADH/NAD+) affinities of the variants. We have investigated four patients with type I RCM, one of whom was homozygous for the D239G mutation. The other three were compound heterozygous: R159-/D239G; G75S/V252M; and P275L/G291D, and one mutation, P275L, was novel. All patients showed reduced enzyme activity, in the range 0.5 to 5.8 IU/g Hb compared to normal values of 7.2 to 26.9 IU/g Hb. Individual variant proteins were prepared and the analytical data are summarised in the Table below.

Recessive congenital methemoglobinemia (RCM, OMIM 250800) arises from defects in either the erythrocytic or microsomal forms of the flavoprotein, cytochrome b5 reductase (cb5r) and was the first disease to be directly associated with a specific enzyme deficiency. Of the 33 verified mutations in cb5r that give rise to either the type I (erythrocytic) or type II (generalized) forms of RCM, three of the mutations, corresponding to P144L, L148P, and R159*, are located in a segment of the primary sequence composed of residues G143 to V171 which serves as a “hinge” or “linker” region between the FAD- and NADH-binding lobes of the protein. With the exception of R159*, which produces a truncated non-functional cb5r resulting in type II RCM, the type I methemoglobinemias resulting from the P144L or L148P mutations have been proposed to be due to decreased enzyme stability. Utilizing a recombinant form of the rat cb5r enzyme, we have generated the P144L, L148P, and P144L/L148P mutants, purified the resulting proteins to homogeneity and characterized their spectroscopic, kinetic, and thermodynamic properties. The three mutant proteins retained full complements of FAD with the P144L and L148P variants being spectroscopically indistinguishable from wild-type cb5r. In contrast, kinetic analyses revealed that the P144L, L148P, and P144L/L148P variants retained only 28, 31, and 8% of wild-type NADH:cytochrome b5 reductase activity, respectively, together with significant alterations in affinity for both NADH and NAD+. In addition, FAD oxidation–reduction potentials were 32, 19, and 65mV more positive for the mutants than the corresponding FAD/FADH2 couple in native cb5r (E0′=-272mV). Thermal and proteolytic stability measurements indicated that all three mutants were less stable than the wild-type protein while differential spectroscopy indicated altered pyridine nucleotide binding in all three variants. These results demonstrate that the “hinge” region is important in maintaining the correct orientation of the flavin- and pyridine nucleotide-binding lobes within the protein for efficient electron transfer and that the P144L and L148P mutations disrupt the normal registration of the FAD- and NADH-binding lobes resulting in altered affinities for both the physiological reducing substrate, NADH and its product, NAD+.

NADH:cytochrome b5 oxidoreductase catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. Utilizing a heterologous expression system for the soluble, catalytic domain of the rat microsomal enzyme, we have produced two mutants, corresponding to E255- and G291D. These mutants correspond to the two specific mutations that were identified over a half century later following diagnosis of the original cases of type I recessive congenital methemoglobinemia (RCM). We have purified both the E255- and G291D variants to homogeneity to determine the molecular basis for type I RCM in these individuals. Both the E255- and G291D variants retained a full complement of FAD and exhibited absorption and CD spectroscopic properties comparable to those of the wild-type protein. Oxidation–reduction potentiometric titrations yielded standard midpoint potentials (E0′) for the FAD/FADH2 couple of −271 and −273 mV for the E255- and G291D variants, respectively, which were comparable to the value of −268 mV obtained for the wild-type protein and confirmed that the redox potential of the flavin was unaffected by either mutation. Thermal and proteolytic stability studies revealed that while the G291D variant exhibited stability comparable to that of wild-type, the E255- variant was markedly less stable, indicative of an altered conformation. Initial-rate kinetic studies revealed that both mutants had decreased catalytic activity (kcat), with the E255- and G291D variants retaining approximately 38 and 58% of wild-type activity, respectively. However, the affinity for NADH (KmNADH) was decreased ∼100-fold for E255- compared to only ∼1.3-fold for G291D, results supported by the spectroscopic binding constant (Ks) obtained for G291D. These results indicate that the properties of both the E255- and G291D cytochrome b5 oxidoreductase mutants are similar to those of other variants that have been identified as resulting in the type I form of RCM.

Cyclic voltammograms were simulated using DigiSim software for reaction mechanisms involving multiple electron transfer steps coupled to proton transfer. Specifically, the overall reaction mechanism of the form O+2e(-)+2H(+) right harpoon over left harpoon RH(2) was used to simulate experimental reduction potentials as a function of pH. The pH-dependent reduction potentials reported in the literature for flavodoxin and free flavin adenine dinucleotide were simulated based on selected reduction potentials and acid dissociation constants. Relationships between reduction potentials and acid dissociation constants are presented to model n=1 and n=2 reaction mechanisms and one- and two-proton-coupled redox reactions. Experimental parameters used in the simulations were selected such that the electron and proton transfer reactions were not rate limiting, and therefore these simulated reactions involve thermodynamic coupling rather than concerted kinetic processes.