Peer leader perspectives from a PLTL implementation in a Hispanic-serving institution.

Peer-Led Team Learning (PLTL) is a pedagogical approach that has been shown to benefit all students, especially underrepresented minority students and peer leaders in Science, Technology, Engineering, and Mathematics (STEM) disciplines. In this work, we present results from our study of the impact of PLTL on our peer leaders from a controlled implementation in general biology, general chemistry, and statistics courses at a Hispanic-serving, minority-serving institution. More specifically, we have measured our PLTL program's impact on our peer leaders' skill development, engagement with the subject material, and sense of belonging as peer leaders. Weekly peer leader reflections analyzed using the Dreyfus model exhibited a consistent set of skills, while those analyzed using the Pazos model revealed a consistent type of student-peer leader interactions, allowing for peer leaders to be assigned to specific levels in the hierarchy of each of the models. Analysis of eight skill-based Likert-scale questions on the SALG survey showed an overall positive shift at the highest level. Independent of the skill or interaction level of the peer leader, we observed several instances of peer leaders acknowledging development in their communication skills, sincere attempts at creating an engaging classroom, and a deep investment in their student's success. Peer leaders also reported improvements in understanding of the subjects they were teaching, wanting to persevere and solve problems independently, and feeling passionate about helping other students.

Screening for Novel Fluorescent Nucleobase Analogues Using Computational and Experimental Methods: 2-Amino-6-chloro-8-vinylpurine (2A6Cl8VP) as a Case Study.

Novel fluorescent nucleic acid base analogues (FBAs) with improved optical properties are needed in a variety of biological applications. 2-Amino-6-chloro-8-vinylpurine (2A6Cl8VP) is structural analogue of two existing highly fluorescent FBAs, 2-aminopurine (2AP) and 8-vinyladenine (8VA), and can therefore be expected to have similar base pairing as well as better optical properties compared to its counterparts. In order to determine the absorption and fluorescence properties of 2A6Cl8VP, as a first step, we used TD-DFT calculations and the polarizable continuum model for simulating the solvents and computationally predicted absorption and fluorescence maxima. To test the computational predictions, we also synthesized 2A6Cl8VP and measured its UV/vis absorbance, fluorescence emission, and fluorescence lifetime. The computationally predicted absorbance and fluorescence maxima of 2A6Cl8VP are in reasonable agreement to the experimental values and are significantly redshifted compared to 2AP and 8VA, allowing for its specific excitation. The fluorescence quantum yield of 2A6Cl8VP, however, is significantly lower than those of 2AP and 8VA. Overall, 2A6Cl8VP is a novel fluorescent nucleobase analogue, which can be useful in studying structural, biophysical, and biochemical applications.

Comparing ultrafast excited state quenching of flavin 1,N6-ethenoadenine dinucleotide and flavin adenine dinucleotide by optical spectroscopy and DFT calculations.

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs. Most of these analogs involve modification of the flavin ring, and there is recent evidence that adenine (Ade)-modified FAD can affect enzyme turnover, but so far this has only been shown for enzymes where the adenine and flavin rings are close to each other in a stacked conformation. FAD is also stacked in aqueous solution, and its photodynamics are quite different from unstacked FAD or FMN. Oxidized photoexcited FAD decays rapidly, presumably through PET with Ade as donor and Fl* as acceptor. Definitive identification of the spectral signatures of Ade∙+ and Fl∙- radicals is elusive. Here we use the FAD analog Flavin 1,N6-Ethenoadenine Dinucleotide (εFAD) to study how different photochemical outcomes depend on the identity of the Ade moiety in stacked FAD and its analog εFAD. We have used UV-Vis transient absorption spectroscopy complemented by TD-DFT calculations to investigate the excited state evolution of the flavins. In FAD*, no radicals were observed, suggesting that FAD* does not undergo PET. εFAD* kinetics showed a broad absorption band that suggests a charge transfer state exists upon photoexcitation with evidence for radical pair formation. Surprisingly, significant triplet flavin was produced from εFAD* We hypothesize that the dipolar (ε)Ade moieties differentially modulate the singlet-triplet energy gap, resulting in different intersystem crossing rates. The additional electron density on the etheno group of εFAD supplies better orbital overlap with the flavin S1 state, accelerating charge transfer in that molecule.

Improving Instructional Fitness Requires Change.

Transmission of information has benefitted from a breathtaking level of innovation and change over the past 20 years; however, instructional methods within colleges and universities have been slow to change. In the article, we present a novel framework to structure conversations that encourage innovation, change, and improvement in our system of higher education, in general, and our system of biology education, specifically. In particular, we propose that a conceptual model based on evolutionary landscapes in which fitness is replaced by educational effectiveness would encourage educational improvement by helping to visualize the multidimensional nature of education and learning, acknowledge the complexity and dynamism of the educational landscape, encourage collaboration, and stimulate experimental thinking about how new approaches and methodology could take various fields associated with learning, to more universal fitness optima. The framework also would encourage development and implementation of new techniques and persistence through less efficient or effective valleys of death.

An Ethenoadenine FAD Analog Accelerates UV Dimer Repair by DNA Photolyase.

Reduced anionic flavin adenine dinucleotide (FADH- ) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV-damaged DNA. The initial step involves photoinduced electron transfer from *FADH- to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD-dependent proteins. The role of this proximity has not been unequivocally elucidated. Some studies suggest that Ade is a radical intermediate, but others conclude that Ade modulates the electron transfer rate constant (kET ) through superexchange. No study has succeeded in removing or modifying this Ade to test these hypotheses. Here, FAD analogs containing either an ethano- or etheno-bridged Ade between the AN1 and AN6 atoms (e-FAD and ε-FAD, respectively) were used to reconstitute apo-PL, giving e-PL and ε-PL respectively. The reconstitution yield of e-PL was very poor, suggesting that the hydrophobicity of the ethano group prevented its uptake, while ε-PL showed 50% reconstitution yield. The substrate binding constants for ε-PL and rPL were identical. ε-PL showed a 15% higher steady-state repair yield compared to FAD-reconstituted photolyase (rPL). The acceleration of repair in ε-PL is discussed in terms of an ε-Ade radical intermediate vs superexchange mechanism.

Roles of subunit NuoL in the proton pumping coupling mechanism of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.

Respiratory complex I has an L-shaped structure formed by the hydrophilic arm responsible for electron transfer and the membrane arm that contains protons pumping machinery. Here, to gain mechanistic insights into the role of subunit NuoL, we investigated the effects of Mg2+, Zn2+ and the Na+/H+ antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on proton pumping activities of various isolated NuoL mutant complex I after reconstitution into Escherichia coli double knockout (DKO) membrane vesicles lacking complex I and the NADH dehydrogenase type 2. We found that Mg2+ was critical for proton pumping activity of complex I. At 2 µM Zn2+, proton pumping of the wild-type was selectively inhibited without affecting electron transfer; no inhibition in proton pumping of D178N and D400A was observed, suggesting the involvement of these residues in Zn2+ binding. Fifteen micromolar of EIPA caused up to ∼40% decrease in the proton pumping activity of the wild-type, D303A and D400A/E, whereas no significant change was detected in D178N, indicating its possible involvement in the EIPA binding. Furthermore, when menaquinone-rich DKO membranes were used, the proton pumping efficiency in the wild-type was decreased significantly (∼50%) compared with NuoL mutants strongly suggesting that NuoL is involved in the high efficiency pumping mechanism in complex I.

Semiquinone intermediates are involved in the energy coupling mechanism of E. coli complex I.

Complex I (NADH:quinone oxidoreductase) is central to cellular aerobic energy metabolism, and its deficiency is involved in many human mitochondrial diseases. Complex I translocates protons across the membrane using electron transfer energy. Semiquinone (SQ) intermediates appearing during catalysis are suggested to be key for the coupling mechanism in complex I. However, the existence of SQ has remained controversial due to the extreme difficulty in detecting unstable and low intensity SQ signals. Here, for the first time with Escherichia coli complex I reconstituted in proteoliposomes, we successfully resolved and characterized three distinct SQ species by EPR. These species include: fast-relaxing SQ (SQNf) with P1/2 (half-saturation power level)>50mW and a wider linewidth (12.8 G); slow-relaxing SQ (SQNs) with P1/2=2-3mW and a 10G linewidth; and very slow-relaxing SQ (SQNvs) with P1/2= ~0.1mW and a 7.5G linewidth. The SQNf signals completely disappeared in the presence of the uncoupler gramicidin D or squamotacin, a potent E. coli complex I inhibitor. The pH dependency of the SQNf signals correlated with the proton-pumping activities of complex I. The SQNs signals were insensitive to gramicidin D, but sensitive to squamotacin. The SQNvs signals were insensitive to both gramicidin D and squamotacin. Our deuterium exchange experiments suggested that SQNf is neutral, while SQNs and SQNvs are anion radicals. The SQNs signals were lost in the ΔNuoL mutant missing transporter module subunits NuoL and NuoM. The roles and relationships of the SQ intermediates in the coupling mechanism are discussed.

Roles of semiquinone species in proton pumping mechanism by complex I.

Complex I (NDH-1) translocates protons across the membrane using electron transfer energy. Two different coupling mechanisms are currently being discussed for complex I: direct (redox-driven) and indirect (conformation-driven). Semiquinone (SQ) intermediates are suggested to be key for the coupling mechanism. Recently, using progressive power saturation and simulation techniques, three distinct SQ species were resolved by EPR analysis of E. coli complex I reconstituted into proteoliposomes. The fast-relaxing SQ (SQ(Nf)) signals completely disappeared in the presence of the uncoupler gramicidin D or the potent E. coli complex I inhibitor squamotacin. The slow-relaxing SQ (SQ(Ns)) signals were insensitive to gramicidin D, but they were sensitive to squamotacin. The very slow-relaxing SQ (SQ(Nvs)) signals were insensitive to both gramicidin D and squamotacin. Interestingly, no SQ(Ns) signal was observed in the ΔNuoL mutant, which lacks transporter module subunits NuoL and NuoM. Furthermore, we sought out the effect of using menaquinone (which has a lower redox potential compared to that of ubiquinone) as an electron acceptor on the proton pumping stoichiometry by in vitro reconstitution experiments with ubiquinone-rich or menaquinone-rich double knock-out membrane vesicles, which contain neither complex I nor NDH-2 (non-proton translocating NADH dehydrogenase). No difference in the proton pumping stoichiometry between menaquinone and ubiquinone was observed in the ΔNuoL and D178N mutants, which are considered to lack the indirect proton pumping mechanism. However, the proton pumping stoichiometry with menaquinone decreased by half in the wild-type. The roles and relationships of SQ intermediates in the coupling mechanism of complex I are discussed.

Semiquinone and cluster N6 signals in His-tagged proton-translocating NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.

NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 µmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.

Excited-state electronic properties of 6-methylisoxanthopterin (6-MI): an experimental and theoretical study.

6-Methylisoxanthopterin (6-MI) is a pteridine-based guanine analog that has a red-shifted absorption and high fluorescence quantum yield. Its Watson-Crick base-pairing and base stacking properties are similar to guanine. The fluorescence quantum yield of 6-MI is sensitive to its nearest neighbors and base stacking, making it a very useful real-time probe of DNA structure. The fundamental photophysics underlying this fluorescence quenching by base stacking is not well understood. We have explored the excited-state electronic structure of the 6-MI in frozen 77 K LiCl glasses using Stark spectroscopy. These measurements yielded the direction and degree of charge redistribution for the S(0)→S(1) transition as manifested in the difference dipole moment, Δµ(01), and difference static polarizability, TrΔα. TDDFT (time-dependent density functional theory) was employed to calculate the transition energy, oscillator strength, and the dipole moments of the ground and lowest optically bright excited state of 6-MI (S(0)→S(1)). The direction of Δµ(01) was assigned in the molecular frame based on the Stark data and calculations. These results suggest that the C4═O and C2-NH(2) groups are electron-deficient in the excited state, a very different outcome compared with guanine. This implies that Watson-Crick hydrogen bonding in 6-MI may be modulated by absorption of a photon so as to strengthen base pairing, if only transiently. Solvatochromism was also obtained for the absorption and emission spectra of 6-MI in various solvents and compared with the Stark spectroscopic results using both the Lippert-Mataga and Bakhshiev models.

Photoinduced electron transfer occurs between 2-aminopurine and the DNA nucleic acid monophosphates: results from cyclic voltammetry and fluorescence quenching.

2-Aminopurine (2AP) is a fluorescent adenine analogue that is useful in part because its substantial fluorescence quantum yield is sensitive to base stacking with native bases in ss- and ds-DNA. However, the degree of quenching is sequence dependent and the mechanism of quenching is still a matter of some debate. Here we show that the most likely quenching mechanism in aqueous solution involves photoinduced electron transfer (PET), as revealed by cyclic voltammetry (CV) performed in aprotic organic solvents. These potentials were used with spectroscopic data to obtain excited-state reduction and oxidation potentials. Stern-Volmer (S-V) experiments using the native base monophosphate nucleotides (NMPs) rGMP, rAMP, rCMP, and dTMP were performed in aqueous solution to obtain quenching rate constants kq. The results suggest that 2AP* can act as either an electron donor or an electron acceptor depending on the particular NMP but that PET proceeds for all NMPs tested.

Differential fluorescence quenching of fluorescent nucleic acid base analogues by native nucleic acid monophosphates.

Fluorescent nucleic acid base analogues (FBAs) are used widely as probes of DNA and RNA structure and dynamics. Of increasing utility are the pteridone adenosine analogues (6MAP, DMAP) and pteridine guanosine analogues (3MI, 6MI). These FBAs (collectively referred to as PTERs) are useful, in part, because their fluorescence quantum yields, Phi(f), are modulated by base stacking with native bases (NBs), making them sensitive reporters of DNA structure. The quenching mechanism has been hypothesized to be photoinduced electron transfer following selective excitation of the FBA, but hard evidence for this has been lacking. The degree of quenching shows some dependence on the neighboring bases, but there has been no real determination as to whether FBA*:NB complexes satisfy the basic thermodynamic requirement for spontaneous PET: a negative free energy for the electron transfer reaction. Indeed, quenching may result from entirely different mechanisms. To address these questions, Stern-Volmer (S-V) experiments were performed using the native-base monophosphate nucleotides (NMPs) GMP, AMP, CMP, and dTMP in aqueous solutions as quenchers to obtain quenching rate constants, k(q). Cyclic voltammetry (CV) and optical absorption and emission data of the PTERS were obtained in aprotic organic solvents. These data were used to obtain excited-state redox potentials from which electron transfer free energies were derived using the Rehm-Weller equation. The reorganization energies for PET were obtained using the Scandola-Balzani equation, taking into account the free energy contribution due to water. 6MAP*, DMAP*, and 3MI* gave negative free energies between -0.1 and -0.2 eV and reorganization energies of about 0.13 eV. They all displayed ET activation energies below the accessible thermal energy (0.038 eV = 3/2k(B)T, where k(B) is Boltzmann's constant) for all NMPs with the exception of CMP, whose activation barrier was only about 35% higher (approximately 0.05 eV). Thus, we conclude that these PTERs act as electron acceptors and promote NMP oxidation. However, 6MI* had positive ET free energies for all NMPs with the exception of GMP (and then only for nucleobase oxidation). The magnitudes of these free energies (> or = 0.45 eV for AMP, CMP, and dTMP) suggest that 6MI* may not quenched by PET.

Change in electronic structure upon optical excitation of 8-vinyladenosine: an experimental and theoretical study.

8-Vinyladenosine (8VA) is an adenosine analog, like 2-aminopurine (2AP), that has a red-shifted absorption and high fluorescence quantum yield. When introduced into double-stranded DNA (dsDNA), its base-pairing and base-stacking properties are similar to those of adenine. Of particular interest, the fluorescence quantum yield of 8VA is sensitive to base stacking, making it a very useful real-time probe of DNA structure. The fundamental photophysics underlying this fluorescence quenching by base stacking is not well understood, and thus exploring the excited state electronic structure of the analog is warranted. In this study, we report on changes in the electronic structure of 8VA upon optical excitation. Stark spectroscopy was performed on 8VA monomer in frozen ethanol glass at 77 K to obtain the direction and degree of charge redistribution in the form of the difference dipole moment, Deltamu(01) = 4.7 +/- 0.3 D, and difference static polarizability, tr(Delta(alpha)01) = 21 +/- 11 A(3), for the S(0)-->S(1) transition. In addition, solvatochromism experiments were performed on 8VA in various solvents and analyzed using Bakhshiev's model. High level ab initio methods were employed to calculate transition energies, oscillator strengths, and dipole moments of the ground and excited states of 8VA. The direction of Deltamu(01) was assigned in the molecular frame for the lowest optically accessible state. Our study shows that the angle between ground and excited state dipole moment plays a critical role in understanding the change in electronic structure upon optical excitation. Compared to 2AP, 8VA has a larger difference dipole moment which, with twice the extinction coefficient, suggests that 8VA is superior as a two-photon probe for microscopy studies. To this end, we have measured the ratio of the two-photon fluorescence yields of the two analogs by excitation at the respective monomer absorption maxima. We show that 8VA is indeed a significantly brighter two-photon fluorophore, based on our experimental and computational results.