Derek McLachlin - Academia.edu (original) (raw)

Papers by Derek McLachlin

The “testing effect” refers to the observation that retrieving information as part of being teste... more The “testing effect” refers to the observation that retrieving information as part of being tested improves retention of information. This phenomenon suggests that a potentially effective study strategy would be for students to generate and respond to their own questions about the material they wish to learn. To this end, two of us (JMM and RAG) created the website CanYouRecall.com, which allows users to enter questions and answers in an open-ended format, and prompts users to answer their own questions after a user-defined interval of time. Allowing users to define how much time has passed between prompts enables them to make use of the “spacing effect”, the observation that memory recall is improved if study is spaced out over a longer rather than a shorter period of time. We conducted a study in a second-year introductory Biochemistry class to assess how students would use CanYouRecall.com , and whether use of the site would improve performance in the course. We hope to repeat the study in Fall 2015 to acquire more participants. This interim presentation will report performance data from the course, comparing students who used the website with those who did not. Marks in first-year Biology courses will be used as a baseline. We will also present an analysis of student participation on the site, the nature of questions that students created using the website, and results of a survey of student opinion about the site

Developing the ability of science students to write is challenging, partly because grading writte... more Developing the ability of science students to write is challenging, partly because grading written work and providing effective feedback is time-consuming. However, the ability to communicate effectively in writing is essential to a university science student’s education. To give our students practice writing at an early stage in our program, we introduced a written assignment into our introductory biochemistry course, which typically has an enrolment of about 1200 students. Expectations for the assignment were communicated through carefully constructed instructions and a simple but detailed rubric. Students uploaded their assignments to the software Crowdmark, which allows instructors to assign TAs to specific submissions, follow their progress, and track time spent by TAs on marking. TAs generated their own comment bank, and assigned grades using instructor-created comments carrying point values such that grades on each assignment were automatically summed. Marks and feedback were returned to students electronically. In this session we will explain how the assignment was designed to develop students’ writing ability and show how Crowdmark was used for grading. The benefits of an online tool for grading many written assignments included easy distribution of assignments to TAs, automatic summing and recording of student marks, and rapid entry of legible feedback

Biochemistry and Molecular Biology Education, 2018

This song alludes to the main sources of stored energy used by the brain in an extended fasting s... more This song alludes to the main sources of stored energy used by the brain in an extended fasting situation: first glycogen, then proteins (broken down to provide starting material for gluconeogenesis), then fatty acids. Notably for the song, because the brain does not directly take up fatty acids, the liver converts fatty acids to ketone bodies, which are then released into the bloodstream for the brain's benefit. The brain can absorb ketone bodies and convert them to acetyl-CoA, allowing ATP production through the Kreb's cycle and oxidative phosphorylation.

Analytical Chemistry, 2003

Protein Expression and Purification, 1996

A method of screening transformed bacterial colonies for introduction of a cysteine residue into ... more A method of screening transformed bacterial colonies for introduction of a cysteine residue into an overexpressed protein is described. After treating SDS extracts of induced bacterial cells with fluorescein-5-maleimide, the proteins containing cysteine were visible on SDS-PAGE gels under ultraviolet light as fluorescent bands. If the wild-type protein contains no endogenous cysteine residues, then mutant proteins containing cysteine may be easily identified by their fluorescence. In addition, a shift in electrophoretic mobility of modified proteins was observed, with mutant proteins containing cysteine at more than one site exhibiting incremental decreases in electrophoretic mobility. This effect permits the detection of cysteine mutations even when endogenous cysteines are present. The described method allows the rapid screening of a large number of transformants.

Journal of Biological Chemistry, 2000

The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to... more The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to act as a stator to hold the ␣ 3 ␤ 3 hexamer stationary relative to the a subunit as the ␥⑀c 9-12 complex rotates. Despite their essential nature, the contacts between b and the ␣, ␤, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b 24-156 , a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F 1 sector or to complete F 1 F 0 was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to ␣ and ␤), and 109 and 110 (to ␣ only). Mass spectrometric analysis of peptide fragments derived from the b 24-156 A92C crosslink revealed that cross-linking took place within the region of ␣ between Ile-464 and Met-483. This result indicates that the b dimer interacts with the ␣ subunit near a non-catalytic ␣/␤ interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F 0 in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b 24-156 A92C and ␤ as well as b 24-156 I109C and ␣ are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex. ATP synthase, or F 1 F 0-ATPase, utilizes a transmembrane proton gradient to synthesize ATP and is responsible for the final step in oxidative phosphorylation and photophosphorylation. The enzyme (reviewed in Refs. 1-3) is composed of two sectors. The membrane-integral F 0 sector is a proton pore, and in Escherichia coli has a subunit composition of ab 2 c 9-12. The membrane-peripheral F 1 sector has a subunit stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. A key feature of the F 1 sector, as seen in the bovine heart mitochondrial crystal structure (4), is that the ␣ and ␤ subunits alternate in a ring around a lengthy pair of ␣-helices of ␥. Each ␤ subunit bears one catalytic nucleotide-binding site, while non-catalytic nucleotide-binding sites are found on the ␣ subunits. These nucleotide-binding sites are located close to the

Journal of Biological Chemistry, 1999

In this study a series of N-and/or C-terminal truncations of the cytoplasmic domain of the b subu... more In this study a series of N-and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F 1 F 0 ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and 15 N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.

Journal of Biological Chemistry, 1997

Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactio... more Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactions in the b subunit of Escherichia coli F 1 F 0-ATPase. Individual cysteine residues were incorporated into b syn , a soluble form of the protein lacking the membrane-spanning Nterminal domain, in two main areas: the heptad repeat region and the hydrophobic region which begins at residue Val-124. The tendencies of these cysteine residues to form disulfide bonds with the corresponding cysteine in the b syn dimer were tested using disulfide exchange by glutathione and air oxidation catalyzed by Cu 2؉. Within the heptad repeat region, only cysteines at residues 59 and 60, which occupy the b and c positions of the heptad repeat, showed significant tendencies to form disulfides, a result inconsistent with a coiled-coil model for b syn. Mixed disulfide formation most readily occurred with the S60C ؉ L65C and A61C ؉ L65C pairs. Cysteines at positions 124, 128, 132, and 139 showed strong tendencies to form disulfides with their mates in the dimer, suggesting a parallel ␣-helical interaction between the subunits in this region. Deletion of residues N-terminal to either Glu-34 or Asp-53 had no apparent effect on dimerization as determined by sedimentation equilibrium, while deletion of all residues N-terminal to Lys-67 produced a monomeric form. These results imply that residues 53-66 but not 24-52 are essential for b syn dimerization. Taken together the results are consistent with a model in which the two b subunits interact in more than one region, including a parallel alignment of helices containing residues 124-139.

Journal of Biological Chemistry, 1998

An affinity resin for the F 1 sector of the Escherichia coli ATP synthase was prepared by couplin... more An affinity resin for the F 1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b 24-156 , a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F 1. Truncated forms of b 24-156 , in which one or four residues from the C terminus were removed, competed poorly for F 1 binding, suggesting that these residues play an important role in b-F 1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b 24-156 resulted in a disruption of its association with the purified ␦ subunit of the enzyme. To determine whether these residues interact directly with ␦, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and ␦ were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b 24-156-F 1 complex and the membranebound F 1 F 0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue ␦ subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and ␦ subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F 1 sector to the b subunit of F 0 .

The Journal of biological chemistry, 2004

We have used site-specific spin-labeling of single cysteine mutations within a water-soluble muta... more We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensio...

Current Opinion in Chemical Biology, 2001

Biochemistry and Molecular Biology Education, 2010

Biochemistry, 2000

... Derek T. McLachlin ‡ and Stanley D. Dunn*. Department of ... Energy-Dependent Fluorescence Qu... more ... Derek T. McLachlin ‡ and Stanley D. Dunn*. Department of ... Energy-Dependent Fluorescence Quenching. The establishment of a proton gradient was monitored by observing the quenching of quinacrine fluorescence essentially as described by Fraga and Fillingame (34). ...

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000

Two stalks link the F 1 and F 0 sectors of ATP synthase. The central stalk contains the Q and O s... more Two stalks link the F 1 and F 0 sectors of ATP synthase. The central stalk contains the Q and O subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic K 3 L 3 complex. The two b subunits and the N subunit associate to form b 2 N, a second, peripheral stalk extending from the membrane up the side of K 3 L 3 and binding to the N-terminal regions of the K subunits, which are approx. 125 A î from the membrane. This second stalk is essential for binding F 1 to F 0 and is believed to function as a stator during rotational catalysis. In vitro, b 2 N is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with N. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one K subunit or KL pair. However, the weak interactions between the polypeptides in b 2 N pose a challenge for the proposed stator function.

Collected Essays on Learning and Teaching, 2011

Music can be used in lectures to increase student engagement and help students retain information... more Music can be used in lectures to increase student engagement and help students retain information. In this paper, I describe my use of biochemistry-related lyrics written to the tune of the theme to the television show, The Flintstones, in a large class setting (400-800 students). To determine student perceptions, the class was surveyed several weeks after the song was used. Students reported a high level of engagement and enjoyment during the song. Many students found the song to be a helpful study tool. To guide future song selection, the students were also asked to indicate their familiarity with 30 popular songs from the past 50+ years. The songs that were least familiar to the students were all released before 1980, but some older songs were well known. The results support the use of content-specific lyrics set to familiar tunes as an educational tool, and provides information about specific songs that would or would not be suitable for this purpose.

Analytical chemistry, Jan 15, 2003

Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues f... more Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues followed by addition of an affinity tag has recently been pursued as a strategy for enriching phosphorylated species from complex mixtures. Here we report the use of an introduced thiol tag as the ligand for affinity purification via disulfide exchange with an activated thiol resin and the development of a protocol to improve the sensitivity considerably over previous reports (i.e., to subpicomole levels.) During our experiments, we observed a side reaction in which water was eliminated from unmodified serine residues. This side reaction resulted in the introduction of the affinity tag into unphosphorylated proteins, confounding attempts to specifically purify phosphoproteins from mixtures. Unchecked, this side reaction will also prevent application of the beta-elimination strategy to phosphopeptide samples where the phosphorylated species are minor components (i.e., most current phosphop...

The Plant Journal, 2002

The development of a germinating embryo into an autotrophic seedling is arrested under conditions... more The development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

The Journal of General Physiology, 2005

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes ... more CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

Journal of Biological Chemistry, 1999

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b sub... more In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.

The “testing effect” refers to the observation that retrieving information as part of being teste... more The “testing effect” refers to the observation that retrieving information as part of being tested improves retention of information. This phenomenon suggests that a potentially effective study strategy would be for students to generate and respond to their own questions about the material they wish to learn. To this end, two of us (JMM and RAG) created the website CanYouRecall.com, which allows users to enter questions and answers in an open-ended format, and prompts users to answer their own questions after a user-defined interval of time. Allowing users to define how much time has passed between prompts enables them to make use of the “spacing effect”, the observation that memory recall is improved if study is spaced out over a longer rather than a shorter period of time. We conducted a study in a second-year introductory Biochemistry class to assess how students would use CanYouRecall.com , and whether use of the site would improve performance in the course. We hope to repeat the study in Fall 2015 to acquire more participants. This interim presentation will report performance data from the course, comparing students who used the website with those who did not. Marks in first-year Biology courses will be used as a baseline. We will also present an analysis of student participation on the site, the nature of questions that students created using the website, and results of a survey of student opinion about the site

Developing the ability of science students to write is challenging, partly because grading writte... more Developing the ability of science students to write is challenging, partly because grading written work and providing effective feedback is time-consuming. However, the ability to communicate effectively in writing is essential to a university science student’s education. To give our students practice writing at an early stage in our program, we introduced a written assignment into our introductory biochemistry course, which typically has an enrolment of about 1200 students. Expectations for the assignment were communicated through carefully constructed instructions and a simple but detailed rubric. Students uploaded their assignments to the software Crowdmark, which allows instructors to assign TAs to specific submissions, follow their progress, and track time spent by TAs on marking. TAs generated their own comment bank, and assigned grades using instructor-created comments carrying point values such that grades on each assignment were automatically summed. Marks and feedback were returned to students electronically. In this session we will explain how the assignment was designed to develop students’ writing ability and show how Crowdmark was used for grading. The benefits of an online tool for grading many written assignments included easy distribution of assignments to TAs, automatic summing and recording of student marks, and rapid entry of legible feedback

Biochemistry and Molecular Biology Education, 2018

This song alludes to the main sources of stored energy used by the brain in an extended fasting s... more This song alludes to the main sources of stored energy used by the brain in an extended fasting situation: first glycogen, then proteins (broken down to provide starting material for gluconeogenesis), then fatty acids. Notably for the song, because the brain does not directly take up fatty acids, the liver converts fatty acids to ketone bodies, which are then released into the bloodstream for the brain's benefit. The brain can absorb ketone bodies and convert them to acetyl-CoA, allowing ATP production through the Kreb's cycle and oxidative phosphorylation.

Analytical Chemistry, 2003

Protein Expression and Purification, 1996

A method of screening transformed bacterial colonies for introduction of a cysteine residue into ... more A method of screening transformed bacterial colonies for introduction of a cysteine residue into an overexpressed protein is described. After treating SDS extracts of induced bacterial cells with fluorescein-5-maleimide, the proteins containing cysteine were visible on SDS-PAGE gels under ultraviolet light as fluorescent bands. If the wild-type protein contains no endogenous cysteine residues, then mutant proteins containing cysteine may be easily identified by their fluorescence. In addition, a shift in electrophoretic mobility of modified proteins was observed, with mutant proteins containing cysteine at more than one site exhibiting incremental decreases in electrophoretic mobility. This effect permits the detection of cysteine mutations even when endogenous cysteines are present. The described method allows the rapid screening of a large number of transformants.

Journal of Biological Chemistry, 2000

The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to... more The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to act as a stator to hold the ␣ 3 ␤ 3 hexamer stationary relative to the a subunit as the ␥⑀c 9-12 complex rotates. Despite their essential nature, the contacts between b and the ␣, ␤, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b 24-156 , a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F 1 sector or to complete F 1 F 0 was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to ␣ and ␤), and 109 and 110 (to ␣ only). Mass spectrometric analysis of peptide fragments derived from the b 24-156 A92C crosslink revealed that cross-linking took place within the region of ␣ between Ile-464 and Met-483. This result indicates that the b dimer interacts with the ␣ subunit near a non-catalytic ␣/␤ interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F 0 in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b 24-156 A92C and ␤ as well as b 24-156 I109C and ␣ are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex. ATP synthase, or F 1 F 0-ATPase, utilizes a transmembrane proton gradient to synthesize ATP and is responsible for the final step in oxidative phosphorylation and photophosphorylation. The enzyme (reviewed in Refs. 1-3) is composed of two sectors. The membrane-integral F 0 sector is a proton pore, and in Escherichia coli has a subunit composition of ab 2 c 9-12. The membrane-peripheral F 1 sector has a subunit stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. A key feature of the F 1 sector, as seen in the bovine heart mitochondrial crystal structure (4), is that the ␣ and ␤ subunits alternate in a ring around a lengthy pair of ␣-helices of ␥. Each ␤ subunit bears one catalytic nucleotide-binding site, while non-catalytic nucleotide-binding sites are found on the ␣ subunits. These nucleotide-binding sites are located close to the

Journal of Biological Chemistry, 1999

In this study a series of N-and/or C-terminal truncations of the cytoplasmic domain of the b subu... more In this study a series of N-and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F 1 F 0 ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and 15 N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.

Journal of Biological Chemistry, 1997

Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactio... more Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactions in the b subunit of Escherichia coli F 1 F 0-ATPase. Individual cysteine residues were incorporated into b syn , a soluble form of the protein lacking the membrane-spanning Nterminal domain, in two main areas: the heptad repeat region and the hydrophobic region which begins at residue Val-124. The tendencies of these cysteine residues to form disulfide bonds with the corresponding cysteine in the b syn dimer were tested using disulfide exchange by glutathione and air oxidation catalyzed by Cu 2؉. Within the heptad repeat region, only cysteines at residues 59 and 60, which occupy the b and c positions of the heptad repeat, showed significant tendencies to form disulfides, a result inconsistent with a coiled-coil model for b syn. Mixed disulfide formation most readily occurred with the S60C ؉ L65C and A61C ؉ L65C pairs. Cysteines at positions 124, 128, 132, and 139 showed strong tendencies to form disulfides with their mates in the dimer, suggesting a parallel ␣-helical interaction between the subunits in this region. Deletion of residues N-terminal to either Glu-34 or Asp-53 had no apparent effect on dimerization as determined by sedimentation equilibrium, while deletion of all residues N-terminal to Lys-67 produced a monomeric form. These results imply that residues 53-66 but not 24-52 are essential for b syn dimerization. Taken together the results are consistent with a model in which the two b subunits interact in more than one region, including a parallel alignment of helices containing residues 124-139.

Journal of Biological Chemistry, 1998

An affinity resin for the F 1 sector of the Escherichia coli ATP synthase was prepared by couplin... more An affinity resin for the F 1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b 24-156 , a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F 1. Truncated forms of b 24-156 , in which one or four residues from the C terminus were removed, competed poorly for F 1 binding, suggesting that these residues play an important role in b-F 1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b 24-156 resulted in a disruption of its association with the purified ␦ subunit of the enzyme. To determine whether these residues interact directly with ␦, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and ␦ were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b 24-156-F 1 complex and the membranebound F 1 F 0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue ␦ subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and ␦ subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F 1 sector to the b subunit of F 0 .

The Journal of biological chemistry, 2004

We have used site-specific spin-labeling of single cysteine mutations within a water-soluble muta... more We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensio...

Current Opinion in Chemical Biology, 2001

Biochemistry and Molecular Biology Education, 2010

Biochemistry, 2000

... Derek T. McLachlin ‡ and Stanley D. Dunn*. Department of ... Energy-Dependent Fluorescence Qu... more ... Derek T. McLachlin ‡ and Stanley D. Dunn*. Department of ... Energy-Dependent Fluorescence Quenching. The establishment of a proton gradient was monitored by observing the quenching of quinacrine fluorescence essentially as described by Fraga and Fillingame (34). ...

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000

Two stalks link the F 1 and F 0 sectors of ATP synthase. The central stalk contains the Q and O s... more Two stalks link the F 1 and F 0 sectors of ATP synthase. The central stalk contains the Q and O subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic K 3 L 3 complex. The two b subunits and the N subunit associate to form b 2 N, a second, peripheral stalk extending from the membrane up the side of K 3 L 3 and binding to the N-terminal regions of the K subunits, which are approx. 125 A î from the membrane. This second stalk is essential for binding F 1 to F 0 and is believed to function as a stator during rotational catalysis. In vitro, b 2 N is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with N. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one K subunit or KL pair. However, the weak interactions between the polypeptides in b 2 N pose a challenge for the proposed stator function.

Collected Essays on Learning and Teaching, 2011

Music can be used in lectures to increase student engagement and help students retain information... more Music can be used in lectures to increase student engagement and help students retain information. In this paper, I describe my use of biochemistry-related lyrics written to the tune of the theme to the television show, The Flintstones, in a large class setting (400-800 students). To determine student perceptions, the class was surveyed several weeks after the song was used. Students reported a high level of engagement and enjoyment during the song. Many students found the song to be a helpful study tool. To guide future song selection, the students were also asked to indicate their familiarity with 30 popular songs from the past 50+ years. The songs that were least familiar to the students were all released before 1980, but some older songs were well known. The results support the use of content-specific lyrics set to familiar tunes as an educational tool, and provides information about specific songs that would or would not be suitable for this purpose.

Analytical chemistry, Jan 15, 2003

Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues f... more Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues followed by addition of an affinity tag has recently been pursued as a strategy for enriching phosphorylated species from complex mixtures. Here we report the use of an introduced thiol tag as the ligand for affinity purification via disulfide exchange with an activated thiol resin and the development of a protocol to improve the sensitivity considerably over previous reports (i.e., to subpicomole levels.) During our experiments, we observed a side reaction in which water was eliminated from unmodified serine residues. This side reaction resulted in the introduction of the affinity tag into unphosphorylated proteins, confounding attempts to specifically purify phosphoproteins from mixtures. Unchecked, this side reaction will also prevent application of the beta-elimination strategy to phosphopeptide samples where the phosphorylated species are minor components (i.e., most current phosphop...

The Plant Journal, 2002

The development of a germinating embryo into an autotrophic seedling is arrested under conditions... more The development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

The Journal of General Physiology, 2005

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes ... more CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

Journal of Biological Chemistry, 1999

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b sub... more In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.