An Interview with Manuel and Ann Varela: John Earnest Walker—A Scientist who was Mentored, Mentored Others and Nobel Prize Winner (with others)

Jul 10, 2020 by

Michael F. Shaughnessy –

1) John Earnest Walker is a British scientist who worked with a Danish scientist and went on to win the Nobel Prize—from very auspicious beginnings. Where was he born in England, when was he born, and where did he initially go to school?

Dr. Sir John Earnest Walker is a chemistry Nobel Laureate. He is renowned for his experimental verification of Paul Boyer’s proposed mechanism for ATP production and the elucidation of the ATP synthase enzyme structure. Walker was born on the 7th day of January, in 1941, in Halifax, West Yorkshire. Walker and his two younger sisters, Judith and Jennifer, grew up in the countryside overlooking the Calder valley near Elland, and then they moved to Rastrick.

Walker’s father, Thomas Ernest Walker, was a stonemason and apparently, a gifted nonprofessional pianist and singer. His mother’s name was Elsie Walker, and the couple had three children. From the ages of 11-18, Walker attended Rastrick Grammar School, which was a Technology College, and graduated with the class of 1960.

2) Often some universities produce great thinkers, researchers, and scholars. Where did he go to university and then medical school?

From 1960 to 1969, Walker was a student at St. Catherine’s College, Oxford, and earned his M.A. in Chemistry. After completing the master’s degree requirements, Walker attended the Sir William Dunn School of Pathology at the University of Oxford, where he studied peptide antibiotics with Edward Abraham from 1965-1969 and took his Ph.D. in 1969. His thesis was entitled Studies on naturally occurring peptides.

From 1969 to 1971, Walker worked at The School of Pharmacy, University of Wisconsin-Madison.

From 1971–1974, Walker was a NATO Fellow at the CNRS, Gif-sur-Yvette, France, and from 1972-1974 he was a Fellow at the Pasteur Institute, Paris, France. He was introduced to Fred Sangerin 1974 at a workshop at the University of Cambridge. This meeting resulted in an offer to work at the Laboratory of Molecular Biology of the Medical Research Council, which became a long-term appointment that ended in 1982. From 1982-1987, Walker was a Senior Scientist at the MRC Laboratory of Molecular Biology, Cambridge. Then, during the years from 1987-1998, Walker had a Special Appointment at the MRC Laboratory of Molecular Biology, Cambridge, where he has remained until retirement as emeritus in 2015.

3) Fred Sanger—a name most scientists know well seems to have had an impact—what did the two study?

According to an autobiography, Walker relates his association with the quite famous Frederick Sanger, who earned two Nobel Prizes. Sanger had taken a chemistry Nobel Prize in 1958 for sequencing the insulin protein and again in 1980 for sequencing DNA.

Walker had first met Sanger in 1974 in Cambridge. Walker had been attending a scientific research workshop on the topic of protein sequence analysis when he first met Sanger during dinner when they were seated together. Walker later told the story that the first thing he said to the famous Nobel Laureate was, “I thought you were dead.”

As the story goes, Walker later called Sanger and inquired whether he had any room in his lab at the MRC for Walker. Sanger was reported to have replied that he had neither funding nor space, but he could join them if he could sort out the funding. Fortunately, Walker had a fellowship that could fund him at Sanger’s lab. Thus, Walker was able to join Sanger’s laboratory at the Molecular Biology Laboratory Medical Research Council (MRC) Laboratory, wherein Cambridge, England, Sanger was housed.

Thus, in 1974 Walker moved to the Protein and Nucleic Acid Division at the MRC. After the funding ran out, Sanger invited Walker to continue his research with Sanger’s group for another year. Walker continued incrementally for additional years, with a renewal of funding each year being provided by Sanger. At the MRC, Walker eventually acquired a tenure-track position.

Walker would later write that Sanger had transformed his scientific career. While it is not entirely clear that Sanger and Walker published together at the MRC—Sanger’s sequencing laboratory was across the hallway from Walker’ s—it was to be, nevertheless, the institution where Walker conducted his Nobel Prize work.

4) Francis Crick—another name that most scientists quickly recognize—and what did the two work together to investigate?

At the MRC, Walker met the inimitable Francis H.C. Crick, 1962 physiology or medicine Nobel Laureate with James Watson and Maurice Wilkins for their accurate elucidation of the DNA structure. Walker would later write that Crick was a member of an elite group of scientists at the MRC who inspired a great deal of excitement and enthusiasm for the pursuit of scientific research. Concerning Crick, who ran his research laboratory in the same building as Walker at the MRC, Walker spoke of the exciting results with Aaron Klug coming out of Crick’s laboratory. Crick and Klug had deduced the molecular structural natures of transfer RNA and chromatin.

5) He was awarded the Nobel Prize along with Jens Skou—but what was his focus in terms of his research contributions?

In 1997, John Walker, Jens Skou, and Paul Boyer shared the chemistry Nobel Prize. As related in the chapter on Skou, he had discovered the sodium-potassium pump. Boyer had deduced a rotational model for the catalytic mechanism during the function of the ATP synthase enzyme. Walker would later test the rotary model by elucidating the molecular structures of the F1 catalytic subunit of the ATP synthase and confirm Boyer’s proposed mechanism.

Walker began his Nobel studies by studying bacteriophages as well as mitochondria of animal cells. He was interested in understanding how energy was produced in these biological systems. Phages are viruses that infect bacteria and exploit the energy-making machinery for its end to make more of the phages. The mitochondria of eukaryotes are organelles that make ATP. Thus, he focused his investigations on ATP synthase.

https://upload.wikimedia.org/wikipedia/commons/5/52/Phage.jpg

https://upload.wikimedia.org/wikipedia/commons/5/52/Phage.jpg

Figure Phages attacking a bacterium

Walker focused his attention, in the late 1970s, on the chemical composition of the ATP synthase enzyme by determining the amino acid sequence of its F1 sub-unit. With Sanger’s laboratory and sequencing expertise nearby, Walker was able to use the latest methods and acquire a good-quality amino acid sequence for the F1 protein. He found that the F1 protein was composed of two smaller peptides, called alpha (α) and beta (β).

Then, using Sanger’s method of sequence comparison analysis, Walker compared the amino acid sequences of the α and β sub-units from his F1 protein with those α and β sequences of F1 molecules from many other organisms. Walker found that particular amino acid sequences were the same in the α and β peptides of F1 sub-units in many completely different organisms. Such identical protein sequences are said to be conserved evolutionarily speaking. Nature requires such conserved sequence motifs for the biological function of proteins, such as, in this case, the energy-making machinery for all cells to live.

Walker found two such conserved protein sequence motifs in the peptides of F1. We know these conserved sequences as Walker A and Walker B motifs, and they are found in the α and the β peptides of the F1 subunits from many organisms. The Walker A motif, which has the amino acids sequence of “Gly-x-x-x-x-Gly-Lys-Thr/Ser.” The Gly denotes the amino acid glycine. Lys is lysine. The four x’s denote any amino acid, and the Thr/Ser designation means that at that position in the protein, a threonine or a serine occupies that location I in the motif. The one-letter code of the motif is “G-x-x-x-x-G-K-T/S.” The Walker A motif is also referred to as the Walker loop, the phosphate-binding loop, or simply P-loop. Thus, the function of the Walker A motif structure is to bind phosphate in proteins that work with ATP or other nucleotides.

The Walker B motif consists of the conserved amino acid sequence of “Arg/Lys-(x)3-Gly-(x)3-Leu-(x)3-Asp,” and the one-letter code is “R/K-(x)3-L-(x)3-D.” Arg (R) denotes arginine. Leu (L) is leucine, and Asp (D) is aspartate. The Walker B sequence has been found to function in ion-binding during ATP hydrolysis.

https://upload.wikimedia.org/wikipedia/commons/8/80/PDB_1ohh_EBI.jpg

https://upload.wikimedia.org/wikipedia/commons/8/80/PDB_1ohh_EBI.jpg

Figure F1 subunit molecular structure—ribbon model

In the mid-1990s, Walker and his collaborator, Dr. Andrew Leslie, used X-ray crystallography to determine the protein molecular structure of the F1 subunit of the ATP synthase enzyme from the cow heart muscle. The new F1 structure determination was a pioneering feat, and the scientific community warmly welcomed it. The view in the figure of the F1 subunit is from the top vantage point, without the accompanying Fo structure.

The protein structure of the F1 subunit itself consists of two smaller peptides, called α and β. There are three copies each of the α and β peptides, making six peptides that constitute the top half of the F1 subunit. The F1 portion also contains a gamma (γ), a delta (δ), and an epsilon (ε) peptide, to make nine total proteins that form the entire F1 segment of the ATP synthase enzyme. Thus, the constitution of an intact F1 structure is written as α3β3γδε.

These nine α3β3γδε peptides of the F1 sub-unit in bovine mitochondria come together to form a massive knob-like structure, reminiscent of a doorknob. These F1 “doorknobs” are visible in electron micrographs and are present along the interior of the mitochondria matrix in animal cells and the cytoplasm of bacteria.

https://upload.wikimedia.org/wikipedia/commons/thumb/0/00/Atp_synthase.PNG/576px-Atp_synthase.PNG

https://upload.wikimedia.org/wikipedia/commons/thumb/0/00/Atp_synthase.PNG/576px-Atp_synthase.PNG

Figure F1 Fo ATP synthase molecular structure – space-filling model

The entire F1 Fo ATP synthase structure, as shown in the figures, sits in the membrane and protrudes outward from the face of the membrane. The F1 subunit has knob dimensions of 10 nanometers in height by 10 nanometers wide. The F1 knob harbors the ATPase enzyme and undergoes the hydrolysis of ATP to form ADP plus phosphate. The Fo subunit consists of three smaller peptides, called a, b, and c. There is one so-called “a” protein, two “b” proteins, and 10-12 “c” proteins that form the Fo subunit. The function of the Fo part of ATP synthase is to reside in the membrane and form a channel for the passage of substrates, like a proton, an ion, or a small molecule.

The entire ATP synthase structure, therefore, has one F1 subunit (with nine peptides: α3β3γδε) and one Fo subunit (with approximately 15 peptides: a1 b2 c10-12). The a and b peptides reside in the membrane adjacent to the 10 to 12 c peptides. The b peptide also has molecular contacts with components of the F1 subunit. In Escherichia coli, the stalk of the ATP synthase is formed by the two b peptides and the delta protein. In some eukaryotes, the stalk is formed by epsilon and gamma peptides.

https://upload.wikimedia.org/wikipedia/commons/3/34/Atpsynthase.jpg

https://upload.wikimedia.org/wikipedia/commons/8/81/Fo_subunit_of_ATPase_C1._Picture_Created_in_PyMol_.png

Figure F1Fo ATP synthase intact structure—on its side—ribbon model

The elucidation of the intact F1Fo ATP synthase structure provided Walker the opportunity to show that the entire machine rotates! The rotatory catalysis hypothesis was first predicted by Paul Boyer and confirmed by Walker as he purified the various conformations of the F1 subunits and showed that they turned around as they made ATP! This rotational means for the ATP synthase energy-making machine permits protons to enter the so-called “a peptide” that is adjacent to the ten or so c subunits of the Fo channel.

https://upload.wikimedia.org/wikipedia/commons/3/34/Atpsynthase.jpg

https://upload.wikimedia.org/wikipedia/commons/3/34/Atpsynthase.jpg

Figure Rotary Catalysis by ATP synthase—arrow shows the direction of rotation

The protons cycle through the channel made by the c subunit collection of the Fo in the membrane and cause the gamma peptide to spin! The protons then exit to the other side of the membrane while facilitating the twirling of the Fo subunit!

Meanwhile, the rotation, in turn, permits the gamma part of the F1 subunit also to rotate. The entire complex machine rotates around the central gamma stalk peptide. The ATP synthase has been clocked to rotate about its axis between 100 and 200 times per second! Therefore, the ATP synthase is an amazing molecular energy-producing machine!

As the rotation proceeds, the ATP is catalytically formed by the alpha and beta peptides. The two alpha and beta peptides of the F1 subunit combine inorganic phosphate with ADP to make ATP. Walker’s work with confirming Boyer’s proposed model for the rotational mechanism of ATP synthesis was the essence of the bestowment of the Nobel to both investigators.

6) Often great scientists spend much time mentoring others—who was lucky enough to have studied under him?

During his career, John Walker was an active research mentor to many postdoctoral fellows under his tutelage. Many of these young scientists moved on to establish productive research programs of their own after receiving their training in Walker’s laboratory.

Among these include J.P. Abrahams, I. Arechaga, M.W. Bowler, S.K. Buchanan, E. Cabezon, I.R. Collinson, H.N. Fernley, N.J. Gay, G. Groth, R. Lutter, B., Miroux, D.P. Narendra, J.L. Rubinstein, M. Saraste, L.A. Sazanov, and D. Stock, among many others.

As of this writing, these investigators are making new advances in the biomedical and biochemical sciences and are considered to be prominent scientists in their own right.

7) He is an Honorary Fellow at St. Catherine’s College in Oxford, England, where one of us (M.F.S.) lectured years ago. What other honors or recognitions were bestowed on him?

Indeed, Walker was widely recognized and bestowed many accolades for his scientific discoveries. A few are mentioned here. In 1995, Walker became a fellow of the Royal Society London. In 1997, Walker, along with Paul Boyer and Jens Skou, was awarded the chemistry Nobel Prize. In 1998, Walker became a founding fellow of the London Academy of Medical Sciences. He has been inducted as a foreign fellow for a series of notational academies such as in The Netherlands, Italy, New Zealand, and the U.S. In 2010, Walker was given the Lee Kong Chian distinguished professorship accolade at the Nanyang Technological University in Singapore.

In 2011, Walker was given the Skou award, granted by Aarhus University in Denmark. In 2012, he received the prestigious Copley medal, sponsored by the Royal Society of London. Another prestigious medal awarded to Walker was that of the Keilin Memorial in 2012 and its associated Lectureship, sponsored by England’s Biochemical Society. In 2019, Sir Walker became an honorary fellow of the prestigious Cambridge Philosophical Society in England.

8) In addition to winning the Nobel Prize—Sir John E. Walker was given a knighthood—what other endeavors did he follow during his later years?

In 1999, Sir John E. Walker was knighted. From 1998-2013, Sir Walker was the Founding Director of the Medical Research Council’s Dunn Human Nutrition Unit in Cambridge. This Unit was later named the MRC Mitochondrial Biology Unit in 2008. As of 2015, Walker has been Emeritus Director and Professor at the MRC Mitochondrial Biology Unit in Cambridge.

9) What have I neglected to ask about Sir John E. Walker?

As a child, he had interests in archeology, flowers, plants, physics, and chemistry. In 1963, Walker married Christina Westcott, and they have two daughters and two granddaughters.

In later years, Walker related in an interview that while in grammar school, he had acquired a lack of self-esteem, and it took many years to overcome it. He attributed the kindness of several teaching and research mentors who had encouraged him as the prime reason for the perseverance and in surmounting his low self-confidence.

It may interest our readers to mention that while in graduate school, one of us (M.F.V.) studied a highly conserved sequence motif, similar to the Walker motifs, in bacteria.

I made mutations in one highly conserved amino acid called glycine of a sequence motif called the Antiporter Motif. This protein sequence motif, also known as Motif C, is found in thousands of secondary active antiporters that are members of the so-called major facilitator superfamily.

I showed that when the conserved glycine was mutated to all other amino acids, only serine or alanine were even remotely acceptable for maintaining the function of resistance to the antibiotic tetracycline in bacteria. All other amino acids in place of the glycine resulted in complete loss of tetracycline resistance. Thus, I was able to establish the functional importance of the Antiporer Motif. In recent times, the motif has been shown to function as a sort of molecular hinge during antibiotic transport across the bacterial membrane.

For additional information about Sir Walker, see the link:

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