Summary of the 1st Bioinorganic Workshop
The first Bioinorganic Workshop, entitled “Trapping Intermediates” was an outreach activity for an NSF grant of Marty Bollinger and Carsten Krebs. It was offered to 36 students and postdocs immediately before the 2010 Frontiers in Metallobiochemistry Symposium. Of the 36 participants, four were foreign (one graduate student each from Japan and Canada, a post-doc from Portugal, and a visiting scientist from Ethiopia) and the remaining 32 were domestic, academic scientists. 34 of the 36 participants stayed for the 2010 Summer Symposium in Molecular Biology, the second “Frontiers in Metallobiochemistry”. This conference allowed the trainees to attend 26 lectures by some of the most distinguished researchers in the field.
The training session had three parts. The first meeting began June 1 in the morning with registration, during which the participants received a folder with meeting materials. These included printed copies of the powerpoint presentations for parts 1 and 3, as well as a DVD containing video clips showing selected important operations that were relevant to the experimental methods of part 2 but were too time consuming to cover within the 90-min time slot for each of the experimental modules. Also during the morning of June 1, the first part of the training session, consisting of a 2 h lecture on stopped-flow absorption and freeze-quench methods (given by Marty Bollinger) and a 2-h lecture on EPR and Mössbauer spectroscopies (given by Carsten Krebs, slides) took place.
The second part was the experimental section, which took place in the afternoon of June 1 and the morning of June 2. The 36 participants were divided into six groups of six people. The groups cycled through four experimental sections: (1) stopped-flow absorption (SF-Abs) spectroscopy, (2) the freeze-quench (FQ) method, (3) EPR spectroscopy, and (4) Mössbauer spectroscopy. Each of the experimental modules lasted 90 min and was offered to each group. For three of the four modules (SF, FQ, and EPR), each group was separated into two subgroups and participants were trained on two different experimental setups. The logistics of the hands-on training required that, at any given time, 24 trainees participated in experiments while the remaining 12 trainees had “bye” rounds. During their “breaks,” the participants had the opportunity to network amongst themselves and with Penn State bioinorganic researchers who were at that point not involved in hosting experiments. This networking was facilitated by offering a poster session concurrent with the experimental modules. Nearly all participants displayed their posters, which they brought for the subsequent “Frontiers in Metallobiochemistry” symposium. Penn State students also displayed posters.
For the SF module, each group was divided into two subgroups of three people, and both SF instruments in the co-PIs’ laboratory were used. After an introduction to the instruments (10 min) one subgroup carried out an experiment on the reaction of the TauD•Fe(II)•aKG•taurine complex with O2 (both unlabeled and deuterated taurine were used to demonstrate the deuterium kinetic isotope effect in the data analysis section). The TauD reaction was monitored at a single wavelength (320 nm), which conveniently reports formation and decay of the Fe(IV)-oxo intermediate. The other subgroup studied the reaction of Mn(II)/Fe(II)-Ct RNR-R2 with O2 with multi-wavelength detection. Each demonstration took 35-40 min, after which the two subgroups switched to see the other SF experiment. In the last five minutes of this module, practical differences between the Bio-Logic SF instrument used in Mike Green’s group and the Applied Photophysics instruments used in the Bollinger/Krebs group were discussed.
For the FQ module, each group was again divided into two subgroups of three participants, and experiments were carried out on two different Update Instruments FQ units. In the first 10 minutes, the technical details of the instrument were described, followed by preparation of mock FQ samples, which the trainees did themselves. Gang Xing demonstrated the preparation of EPR samples using the more commonly used cryosolvent, isopentane, while Courtney Krest demonstrated the preparation of Mössbauer samples using liquefied ethane as the cryosolvent. Because the Update Instruments FQ unit is no longer commercially available, the use of an alternative (still available) instrument from Bio-Logic was also demonstrated in the last ten minutes of this experimental module.
For the EPR module, the group was again divided into two subgroups, and use of two different spectrometers was demonstrated. Post-doc Wei Jiang used our old “workhorse” X-band EPR spectrometer, while Mike Green used the pulsed X-band spectrometer available to the bioinorganic group. A 10-min introduction to the basic working principles of, and safety matters associated with, EPR was first presented, followed by demonstration of parameter settings, data collection, data manipulation, and spin quantification of the Mn(IV)/Fe(IV) intermediate of Ct R2 by comparison to the spectrum of a Cu(II) standard. The determination of the “packing factor” (the dilution factor associated with the FQ technique due to the presence of the cryosolvent in the sample) was also demonstrated. Mike Green offered a brief introduction showing how to use the free software, EasySpin, for data analysis and spectral simulation.
The Mössbauer module was led by Becky Keller and Carsten Krebs. During the 90-min tutorial, two spectra of intense samples were collected for ~ 40 min each. The first was of a calibration standard, α-Fe foil, and the second of the stable Mn(IV)/Fe(III) cluster in Ct RNR-R2. During data collection, the configuration of the spectrometers and principles of data analysis were explained. The session concluded with the analysis of the spectrum of the Mn(IV)/Fe(III) cluster in Ct RNR-R2 collected by the group.
The third and last section of the training program involved manipulation and analysis of the data collected during the practical sessions (as well as some additional related data). This part was conducted in a lecture hall and carried out on multiple computers that projected to a screen. It was mostly administered by graduate students and post-docs of the Bollinger/Krebs group. Key figures of our published papers on the two model enzyme systems were reconstructed step-by-step during this session.