My Mentor - Prof. Alfred Guillou Redfield
Prof. Tai-huang Huang
I decided to major in biophysics after passing Ph.D. qualifying examination in Physics at Brandeis University in 1973. I was lucky that Prof. Redfield accepted me as his first graduate student at Brandeis. As a new comer in biophysics himself, Prof. Redfield knew the importance of biology in my future career as a biophysicist. He put me through a series of courses, starting from the most fundamental cell and molecular biology to biochemistry, molecular biology, and structural biology etc. These courses were rather challenging for a physicist not yet fluent in English. I truly appreciate Prof. Redfield’s patient and meticulous attention in nurturing me through this period of time.
As described in the obituary written by Prof. Tom Pochapsky and Prof. Mary Roberts, Prof. Redfield made seminal contributions to both theoretical and experimental aspects of nuclear magnetic resonance. Yet, Prof. Redfield is a very low-key and humble person. He had a wet lab in Biochemistry and an electronic lab in Physics and kept a relatively moderate size group. As I recall, the most he ever had was two postdoctoral fellows, two graduate students, one visiting scholar, one hardware associate, and one web lab technician. Probably running under tight budget Prof. Redfield often took students and postdocs to conferences by his square VW minibus, even to as far as Atlantic City in New Jersey, which is a good six-hour drive. Once he drove us to a meeting in Albany, N.Y. To our embarrassment, Prof. Redfield reserved only one hotel room and he insisted on sleeping on sofa while postdoc and I slept on beds at night. He liked to tell a story that when on a sabbatical leave in Daniel Koshland’s lab at Berkeley he tried to use the NMR spectrometer and was asked by a student whether he knew how to use it. The student thought he was a janitor since he dressed so plainly. Prof. Redfield was a hands-on, hard working person. In addition to building his own spectrometer and wrote the processing software himself Prof. Redfield even worked in the wet lab and purified his own sample. I remember once saw him carrying a 2-liter jar of E. coli cells culture walking down the hall way of the Biochemistry building late in a cold winter night.
Benefited from the availability of high-field superconducting magnet and the ever-increasing power of computer, NMR underwent rapid development during the late 1960s. It became feasible for NMR to probe macromolecular structure and function. However, it was still a daunting task to observe high quality NMR spectrum of macromolecules. The first hurdle to be overcome was the 105 fold dynamic range problem when detecting protein signal at millimolar concentration in the presence of 110 M water signal. To overcome this problem, Prof. Redfield invented the “2-1-4” pulse sequence technique which is the conceptual basis for others to develop the more sophisticated WATERGATE and other composite pulse sequences.
“2-1-4” referred to a strain of 90° pulses of proper phases and pulse widths 2τ,τ, 4τ,τ, 2τ, where τ = 1/Δν and Δν is the spectral bandwidth. By placing the reference frequency at the center of interested spectral region and set water resonance at the edge “2-1-4” pulse train generates a relatively flat power spectrum over 80% of the observation window and with water signal largely suppressed. As to be expected, the excitation power spectrum was not a perfect square shape and the signal had significant amplitude and phase distortions near the edges of the observation window. To correct the imperfection Prof. Redfield generated an array of look-up tables containing the distortion information under different setting. The spectrum was corrected accordingly with his processing program on a NOVA 1220 computer. Pulse lengths, amplitudes and phases were set and tweaked manually with knobs. The signal was detected in quadrature mode, a novel idea introduced by Prof. Redfield that enhances the sensitivity by a factor of 1.414. Spectra were normally obtained at relatively low resolution. Prof. Redfield preferred to show non-massaged discrete ladder-type spectra. Another interesting feature of the spectrometer is the shimming. Free induction decay (FID) signals were sent to an audio amplifier system and the shimming quality was conveniently judged by the length of the audio ringing without visualizing the FID signal on the screen. The spectrometer performed perfectly with high flexibility and little down time.
Prof. Redfield was a person with few words. He assigned me a thesis topic aimed at elucidating the molecular mechanism of hemoglobin (Hb) cooperativity in oxygen binding by NMR. From there on you are on your own. Hb is a two-way respiratory carrier, transporting oxygen from the lung to the tissue and facilitate the return transport of CO2. It fulfils this role with high efficiency by reversible structural changes that resulted in cooperative oxygen binding to the four α2β2 subunits. In nature, mainy molecules utilizes cooperativity for efficiently carrying out a wide range of functions including transport, catalysis, and signal transduction.
Hb is the gold standard model system for studying the molecular mechanism of cooperativity. My goal was to determine the oxygen binding kinetics at every intermediate steps of the oxygen binding cycle and correlated them to the conformational changes. Such molecular information is vital for discriminating whether cooperativity occurs through a two-state (MWC) model or a sequential (KFN) model.
The binding kinetic data would be determined by the saturation transfer techniques that Prof. Redfield successfully employed to dissect the electronic transfer process in cytochrome c. I purified hemoglobin from blood mainly from myself and from the local hospital. Prof. Redfield also voluntarily contributed several times.
In order to observe protein signal of various oxygenated species, including deoxygenated, mono, - di-, tri-, and fully oxygenated species, I designed an insert system that allowed me to accurately adjust and measure the degree of oxygenation of Hb in the NMR tube. Subsequently, NMR spectrum of the same sample with desired degree of oxygenation can be obtained. We could accurately determine the amount of deoxygenated HB and fully oxygenated Hb species in the sample from the respective markers of these two species.
To my disappointment, we could not detect signal that can be attributed to intermediately oxygenated species. Apparently the fraction of intermediate species were present too low to be detected due to the high degree of cooperativity in oxygen binding to Hb. Without the markers for intermediately oxygenated species, I could not measure the exchange rates among them and my thesis objective could not be achieved. I am sure Prof. Redfield was also disappointed as well, but he allowed me to continue exploring other topics. I was able to determine the relative oxygen binding affinity of α and β chains separately in the intact Hb tetramer. This resulted in my first publication in JBC in 1976.
Subsequently, I published my second JBC paper on the quaternary structure and heterogeneity of nitrosyl- and met-hemoglobin. Notably, Prof. Redfield did not want to have his name on this paper and I was the single author on this paper.
Additionally, I acquired substantial amount of transient saturation NOE data aimed to assigning various resonances of individual α and β chains which I separated from intact Hb teramer. These were taken before the transient NOE experiments become popular. In attempting to assign the resonance from NOE data Prof. Redfield took me to Prof. Furie’s lab at Tufts University to visualize Hb structure on their computer.
He also sent me to Prof. Chien Ho’s lab at Carnegie University and Prof. Winslow Caughey’s lab at Colorado State University to examine their 3D models of oxy- and deoxy-hemoglobin. Unfortunately, the protein was way too big and I was not confident enough to reach definitive assignments from the one-dimensional proton NOE data alone.
Retrospectively, I probably should have focused on obtaining more data for a methodology paper, which would have been the first transient NOE paper in biological system. Although Prof. Redfield might have lost interest in the hemoglobin project after I failed to obtain kinetic data, he continued to provide me strong supports. He brought me to discuss my project with Prof. Martin Karplus’s group at Harvard University. He also introduced me to discuss with Prof. Max Peruz, a Nobel laureate for elucidating the structure and function of hemoglobin. Upon his return to UK, Prof. Peruz wrote me a letter requesting data from me. These are memorable moments in my graduate career. All these arrangements typify Prof. Redfield’s personality that he may appear not very affectionate but deep in his heart he is a warm and caring person.
Prof. Redfield’s interest in field cycling dated back to the early 1960 when he first applied field cycling technique to investigate impurity in copper (Phys. Rev 1963). I also heard from him several times back in the 1970s of investigating field-dependent NMR phenomena by fast shuttling sample to different positions of a superconducting magnet where the stray fields are lower. Apparently he never got the time to do it until his retirement in 1999. Prof. Redfield published his first mechanical design version in Magn. Reson. Chem. in 2003 and the improved pneumatic version in JBNMR in 2012. Prof. Redfield’s field cycling design opened up a door for investigating full-range relaxation of individual nuclear spins of biopolymers with high resolution and sensitivity. The potential was elaborated in his 2012 JBNMR paper and well demonstrated in the series of papers on dissecting membrane lipid structure and dynamics with Prof. Mary Roberts, and on SARS nucleocapsid protein with Prof. Dorothee Kern (Brandeis). Prof. Redfield was rather proud of his idea and happily told me that he received a grant on the field cycling project at the age of 80.
Prof. Redfield attempted to pursuit me to build the shuttling system several times. As much as I appreciated the power of such a technique I did not have the proper personnel capable of building the device until the arrival of Chingyu Chou, a talented physics graduate student who took up the challenge to work on field cycling as her Ph.D. thesis subject. Prof. Redfield sent us the detailed hand-drawings of all parts of his newer pneumatic version of sample shuttler that employs a servomotor-driven rod to rapidly moving the sample up/down to precise locations inside the magnet.
In order to have a first-hand understanding of the design and operation, I also visited Prof. Redfield’s lab at Brandeis University. He spent couple hours or so to show me the assembly and operation of the device and carefully answered all questions I had. With the blue prints at hand, Chingyu quickly built a working shuttler.
Apparently Chingyu was not satisfied and she came out with a clever new design that replace the somewhat tall and bulky servomotor-rod assembly that needs to be wheeled in near the magnet. Chingyu’s new design consists of a servomotor and a pulley/timing belt-drive, a rail inserted inside the center of the magnet for guiding the shuttle, a light-weight acrylic shuttle/sample assembly; and the control electronics interfaced to the spectrometer PC and the spectrometer pulse program (Chou et al. J. Magn. Reson. 2012). Chingyu built and install a second unit for a 700 MHz system with cryoprobe at CEA in France. This setup was employed it to investigate the field-dependent relaxation of MRI contrast agents successfully (Chou et al. Sci. Rep. 7:44770, 2017). Prof. Redfield liked Chingyu’s new design and recommended readers of his 2012 ＪBNMR paper to seriously considering Chingyu’s design when building a fast sample shuttler. Nonetheless, it is a pity that seventeen years has passed since Prof. Redfield published his shuttle design and yet the NMR community has not fully embrace the technology. Probably because the new generation of NMR spectroscopists are primarily commercial instruments users and are not used to build hardware. I am glad that Chingyu continues to promote the technology by establishing the Field-Cycling Technology Ltd. She has since installed two additional systems in Germany (MPI) and USA (NYSBC). It is my wish that readily available of the device will help spread the seeds that Prof. Redfield planted. Chingyu is now carrying on her shoulder the responsibility of continuing Prof. Redfield’s legacy in field cycling technology.