history of science

Functional Promiscuity: The Choreography and Architecture of the Zinc Gang

By Contributing Editor Nuala F. Caomhánach

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Zinc Finger DNA Complex, image by Thomas Splettstoesser

 

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Andreas Sigismund Marggraf

The tale about gag knuckles, taz-two, hairpin, ribbons, and treble clef is quite elusive.  Although they sound more like nicknames of a 1920’s bootlegging gang (at least to me) they are the formal nomenclature of a biochemical classification system, known commonly as zinc fingers (ZnF). The taxonomy of zinc fingers describes the morphological motif that the element creates when interacting with various molecules. Macromolecules, such as proteins and DNA, have developed numerous ways to bind to other molecules and zinc fingers are one such molecular scaffold. Zinc, an essential element for biological cell proliferation and differentiation, was first isolated in 1746 by the German chemist Andreas Marggraf (1709-–1782). Zinc fingers were important in the development of genome editing, and while CRISPR remains king, zinc is making a comeback.

 

Strings of amino acids fold and pleat into complex secondary and tertiary structures (for an overview, see this video from Khan Academy).

 

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Fig. 1: The folding funnel

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Fig. 2: The energy landscape

Proteins with zinc finger domains—meganucleases—act as molecular DNA scissors, always ready to snip and organize genetic material. The return of these biochemical bootleggers, an older generation of genome editing tools, is due to the problem of exploring the invisible molecular world of the cell. In this age of genomic editing, biologists are debating the concept of protein stability and trying to elucidate the mechanism of protein dynamics within complex signalling pathways. Structural biologists imagine this process through two intermingled metaphors, the folding funnel (fig. 1) and the energy landscape (fig. 2). The energy landscape theory is a statistical description of a protein’s potential surface, and the folding funnel is a theoretical construct, a visual aid for scientists. These two metaphors get scientists out of Levinthal’s Paradox, which argues that finding the native or stable 3-dimensional folded state of a protein, that is, the point at which it becomes biologically functional, by a random search among all possible configurations, can take anywhere from years to decades. Proteins can fold in seconds or less, therefore, biologists assert that it cannot be random. Patterns surely may be discovered. Proteins, however, no longer seem to follow a unique or prescribed folding pathway, but move in different positions, in cellular time and space, in an energy landscape resembling a funnel of irregular shape and size. Capturing the choreography of these activities is the crusade of many types of scientists, from biochemists to molecular biologists.

 

Within this deluge of scientific terminology is the zinc atom of this story.  With an atomic number of 30, Zinc wanders in and out of the cell with two valence electrons (Zn2+). If Zinc could speak it might tell us that it wishes to dispose, trade, and vehemently rid-itself of these electrons for its own atomic stability. It leads with these electrons, recalling a zombie with outstretched arms. It is attracted to molecules and other elements as it moves around the cytoplasm and equally repelled upon being cornered by others. Whilst not as reactionary as the free radical Hydrogen Peroxide (H2O2), it certainly trawls its valency net in the hope of catching a break to atomic stasis, at least temporarily. In this world of unseen molecular movements a ritual occurs as Zinc finds the right partner to anchor itself to. Zinc trades the two electrons to form bonds making bridges, links, ties and connections which slowly reconfigure long strings of amino or nucleic acids into megamolecules with specific functions. All of this occurs beneath the phospholipid bilayer of the cell, unseen and unheard by biologists.

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Model of a Zinc atom

The cell itself is an actor in this performance by behaving like a state security system, as it monitors Zn2+ closely. If the concentration gets too high, it will quarantine the element in cordoned-off areas called zincosomes. By arranging chains of amino acids, such as cysteine and histidine, close to each other, the zinc ion is drawn into a protein trap, and held in place to create a solid, stable structure. They connect to each other via a hydrogen atom, with hydrogen’s single electron being heavily pulled on by Carbon as if curtailing a wild animal on a leash. In the 1990s, when the crystal structures of zinc finger complexes were solved, they revealed the canonical tapestry of interactions. It was notable that unlike many other proteins that bind to DNA through the 2-fold symmetry of the double helix, zinc fingers are connected linearly to link sequences of varying lengths, creating the fingers of its namesake. Elaborate architectural forms are created.

Like a dance, one needs hairpins, or rather beta-hairpins, two strands of amino acids that form a long slender loop, just like a hairpin. To do the gag knuckle one needs two short beta-strands then a turn (the knuckle), followed by a short helix or loop. Want to be daring, do the retroviral gag knuckle. Add one-turn alpha-helix followed by the beta hairpin. The tref clef finger looks like a musical treble clef if you squint really hard. First, assemble around a zinc ion, then do a knuckle turn, loop, beta-hairpin and top it off with an alpha-helix. The less-complex zinc ribbon is more for beginners: two knuckle turns, throw in a hairpin and a couple of zinc ions. Never witnessed, biologists interpret this dance using x-ray crystallography, data that looks like redacted government documents, and computer simulated images.

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X-ray crystallography of a Zinc Finger Protein, image from Liu et al., “Two C3H Type Zinc Finger Protein Genes, CpCZF1 and CpCZF2, from Chimonanthus praecox Affect Stamen Development in Arabidopsis,” Genes 8 (2017).

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Aaron Klug

In the 1980s, zinc fingers were first described in a study of transcription of an RNA sequence by the biochemist Aaron Klug. Since then a number of types have been delimited, each with a unique three-dimensional architecture. Klug used a model of the molecular world that required stasis in structure.The pathway towards this universal static theoretical framework of protein functional landscapes was tortuous. In 1904, Christian Bohr (1855–1911), Karl Albert Hasselbalch (1874-1962), and August Krogh (1874-1949) carried out a simple experiment. A blood sample of known partial oxygen pressure was saturated with oxygen to determine the amount of oxygen uptake. The biologists added the same amount of CO2 and the measurement repeated under the same partial oxygen pressure. They described how one molecule (CO2) interferes with the binding affinity of another molecule (O2) to the blood proteins, haemoglobin. The “Bohr effect” described the curious and busy interactions of how molecules bind to proteins.

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Jacques Monod and François Jacob, image by Agence France-Presse

In 1961, Jacques Monod and Francois Jacob extended Bohr’s research. The word ‘allosteric’ was introduced by Monod in the conclusion of the Cold Spring Harbor Symposium of 1961. It distinguished simultaneously the differences in the structure of molecules and the consequent necessary existence of different sites across the molecule for substrates and allosteric regulators. Monod introduced the “induced-fit” model (proposed earlier by Daniel Koshland in 1958). This model states that the attachment of a particular substrate to an enzyme causes a change in the shape of the enzyme so as to enhance or inhibit its activity. Suddenly, allostery erupted into a chaotic landscape of multiple meanings that affected contemporary understanding of the choreography of zinc as an architectural maker. Zinc returned as the bootlegger of the cellular underworld.

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The “induced-fit” model

Around 2000, many biologists were discussing new views on proteins, how they fold, allosteric regulation and catalysis. One major shift in the debate was the view that the evolutionary history of proteins could explain their characteristics. This was brought about by the new imaginings of the space around the molecules as a funnel in an energy landscape. When James and Tawfik (2003) argued that proteins, from antibodies to enzymes, seem to be functionally promiscuous, they pulled organismal natural selection and sexual selection theory into the cellular world. They argued that promiscuity is of considerable physiological importance. Bonding of atoms is more temporal and more susceptible to change than thought of before. Whilst they recognized that the mechanism behind promiscuity and the relationship between specific and promiscuous activities were unknown they opened the door to a level of fluidity earlier models could not contain. Zinc fingers, thus, played an important role in being a “freer” elements with the ability to change its “mind”.

These ideas were not new. Linus Pauling proposed a similar hypothesis, eventually discarded as incorrect, to explain the extraordinary capacity for certain proteins (antibodies) to bind to any chemical molecule. This new wave of thinking meant that perhaps extinct proteins were more promiscuous than extant ones, or there was a spectrum of promiscuity, with selection progressively sifting proteins for more and more specificity. In 2007, Dean and Thornton, reconstructed ancestral proteins in vitro and supported this hypothesis.  If the term promiscuity as it has been placed on these macromolecules sticks, what exactly does it mean?  If promiscuity is defined as indiscriminate mingling with a number of partners on a casual basis, is zinc an enabler? A true bootlegger? This type of language has piqued the interest of biologists who see the importance of this term in evolution and the potential doors it opens in biotechnology today. Promiscuity makes molecular biology sexy.

To state that a protein, or any molecule, is not rigid or puritanical in nature but behaves dynamically is not the equivalent of stating that the origin of its catalytic potential and functional properties has to be looked for in its intrinsic dynamics, and that the latter emerged from the evolutionary history of these macromolecules.  The union of structural studies of proteins and evolutionary studies means biologists have not only (re)discovered their dynamics but also highlights the way that these properties have emerged in evolution. Today, evolutionary biologists consider that natural selection sieves the result, not necessarily the ways by which the result was reached: there are many different ways of increasing fitness and adaptation. In the case of protein folding with zinc fingers, what is sieved is a rapid, and efficient folding choreography. These new debates suggest that what has been selected was not a unique pathway but an ensemble of different pathways, a reticulating network of single-events, with a different order of bond formation.

If the complex signalling pathways inside of a cell begins with a single interaction, zinc plays a star role. Zinc, along with other elements such as Iron, are the underbelly of the molecular world. Until captured and tied down, the new view of proteins offers to bond mechanistic and evolutionary interpretations into a novel field.  This novelty is a crucial nuance to explain the functions and architecture of molecular biology. As the comeback king, zinc fingers are used in a myriad of ways. As a lookout, biologists infected cells with with DNA engineered to produce zinc fingers only in the presence of specific molecules. Zinc reports back as biologist looks for the presence of a molecule, such as a toxin, by looking for the surface zinc fingers rather than the toxin itself. Zinc fingers are obstructionists. In 1994 an artificially-constructed three-finger protein blocked the expression of an oncogene in a mouse cell line. As informants, they capture specific cells.  Scientists have experimented by creating a mixture of labeled and unlabeled cells and incubated them with magnetic beads covered in target DNA strands. The cells expressing zinc fingers were still attached to the beads. They introduce outsiders into the cell, such as delivering foreign DNA into cells in viral research. In tracking these bootleggers, scientists have pushed the limits of their own theories and molecular landscapes, but have landed on promiscuity, a culturally laden, inherently gendered word.

“To seek God in all things”: The Jesuit encounter with botany in India

By contributing writer Joseph Satish V

Only a month after India gained independence from the British in 1947, the Indian botanist Debabrata Chatterjee wrote of his

hope that in the new India the Government will… effect among other things the early revival of the Botanical Survey of India. If it is possible to recruit men of knowledge and qualities of those giants of the past… no man of science in India need doubt that the revival of the Survey would be of the greatest help and of far-reaching benefits to India.

In 1954, the first independent Government of India appointed the taxonomist Hermegild Santapau as its Chief Botanist and Director.  Santapau had a PhD in Botany from the London University, had worked at the Royal Botanical Gardens in Kew (England), was a Fellow of the Linnean Society of London, editor of the Journal of the Bombay Museum of Natural History, and a Professor of Botany. His services to reviving botany and science education in the country were recognized with the award of the Padma Shri from the Government of India. But this “giant” was neither British nor Indian — he was a Catholic Jesuit priest from Spain. Fr. Santapau was only one of the many Jesuits who established a legacy of “Jesuit science” in India after the religious order was “restored” in 1814.

Ignatius at the River Cardoner - By Dora Nikolova Bittau in Chapel of St Ignatius, Seattle University

Ignatius at the River Cardoner .By Dora Nikolova Bittau, in the Chapel of St. Ignatius, Seattle University

The Society of Jesus is a religious congregation of Catholic clerics founded by Ignatius of Loyola (1491-1556) in 1540. Only two years later, Francis Xavier (1506-1552), one of Ignatius’ first companions, reached Goa on the western coast of India. When Ignatius died sixteen years later, the number of Jesuits had grown to a thousand members around the world. With a unique “way of proceeding“, the Jesuits established themselves firmly in secular culture — arts, astronomy, anthropology, even naval architecture — all “for the greater glory of God“. However, the growing influence of the Jesuits in the Church, State, and society caused resentment in many of Catholic Europe’s nations (chiefly Portugal, Spain, and France), which led to the Suppression of the Jesuits by Pope Clement XIV in 1773. Forty-one years later though, Pope Pius VII restored the Society in 1814.

Scholarship in the history of the Jesuits has witnessed a significant shift in the past few decades. Since the 1980s, the number of non-Jesuit (also non-Catholic) scholars interrogating what has come to be called “Jesuit science” has increased. Historians of Jesuit science have generally explored the relationship between the Jesuit missionary goals and their scientific activity in the sixteenth and seventeenth centuries, often portraying the Jesuits as ‘transmitters’ of European science to the colonies in the New World. Steven J. Harris’s description of early modern Jesuit science continues to be used as a universal description of Jesuit science across space and time. But some scholars have begun arguing the case for a more nuanced engagement with regional variants of Jesuit science.

Dhruv Raina explains that the European Jesuits who arrived in South Asia not only transmitted European science but also discovered, collected, and interpreted indigenous knowledge in the colonies. Agustin Udias argues for a broader exploration of Jesuit science in the post-Restoration period beyond Europe. It is worth exploring, even briefly, why post-1814 Jesuit science is considered different in comparison to early modern Jesuit science.

After 1814, the “new” Jesuits of the restored Society found themselves in an alien scientific landscape, which, among other things, was characterized by the emergence of specialized disciplines and “professionals” who were paid to pursue science. Subsequently, the Jesuits reinvented themselves as a teaching order in schools and colleges across the world. But they were forced to shift from the eclectic scientific tradition of their past to the new disciplines like seismology in North America and the “new botany” (laboratory-based botany research) in England and Germany. It was also in this period that the Jesuits returned to India – a group of Belgian Jesuits came to the eastern coast and set up the Bengal Mission in 1834. Later in 1837, the French Jesuits established the Madurai Mission in southern India. While the sixteenth-century Jesuits interacted with Hindu kings and Mughal emperors, now the Jesuits were obligated to cooperate with the British Empire. However, one feature remained common to the scientific enterprise of the “old” and the “new” Jesuits in India: collecting plant specimens.

Harris notes that medical botany – identifying local plants and their benefits for health reasons – was fairly consistent across all the early Jesuit missions. In India, the Jesuits in early modern Goa acquainted themselves with the native medical traditions – the Portuguese Jesuit Garcia de Orta (1500-1568) provided the first instance of the exchange between European and Ayurvedic medical systems. The early Jesuits to India lived as pilgrims, moving between villages and kingdoms, and evangelized the natives. In the process the Jesuits gained knowledge about the local customs, including that of native plants which they consumed as food or medicine. The restored Jesuits were no longer evangelizers but educators of the evangelized. They established training houses (novitiates) for teaching candidates for the priesthood (novices) in subjects that included philosophy, theology as well as the natural and physical sciences. Training in the natural sciences included collecting, identifying and preserving different flora and fauna. Yet, the focus on nature and the sciences was not only necessitated by the educational mission of the Jesuits; it was an integral part of their “spiritual” training.

Ignatius of Loyola believed that one could experience God in the natural world. He writes in his autobiography that he had spiritual experiences while gazing at nature, be it the stars in the night sky or the Cardoner river in his native Spain. Ignatius maintained notes of these experiences, reflected upon them, and later felt that “some things which he used to observe in his soul and found advantageous could be useful also to others, and so he put them into writing”. This took the form of a series of contemplative exercises called the Spiritual Exercises which later became the foundation for the compulsory spiritual training of the Jesuit novices and continues to be so.

The goal of the Spiritual Exercises was (and is) to help the Jesuit to identify his vocation in life. Guided by a spiritual director in solitude, the novice was urged “to use his senses, particularly sight to fix their mental gaze upon the scene of the meditation” during each exercise. Following this, the novice was expected to write down notes of his contemplative experience, like Ignatius did, and maintain an “observational” record of his spiritual experiences. The acme of the exercises was the ‘Contemplation to Attain Love‘ in the Fourth Week where the novice was asked to consider: “… how God dwells in creatures; in the elements, giving them existence; in the plants, giving them life; in the animals, giving them sensation; in human beings, giving them intelligence …” This tradition of contemplating “how God dwells in” nature remained unchanged in the restored Society. This along with an emphasis on silence and solitude encouraged Jesuits to establish their formation houses amidst pristine natural habitats. It was for this reason that the French Jesuits established their novitiate in Shembaganur (1877) close to Kodaikanal, a south Indian hill station favored by the British.

Hand painted plate by Anglade 1919 - Courtesy Rapinat Herbarium Trichy

Hand painted plate by Anglade, 1919. Courtesy Rapinat Herbarium Trichy

Less than a decade before the Shembaganur novitiate was established, the British taxonomist Joseph Dalton Hooker had completed his botanical expedition in the Himalayas and published several illustrated flora (1871). The Royal Botanical Gardens at Kew outside London became “the center of a worldwide network of colonial gardens”. Acknowledging these developments, the French Jesuits (who had already received some training in the natural sciences in Europe) promoted the teaching and learning of the biological sciences at Shembaganur. A part of the novice’s education also included plant taxonomy; the novices had to venture into the nearby Palni hills to identify and collect plant specimens. The young novices had several Jesuits to guide and inspire them. Pierre Labarthere (1831–1904)  cultivated botanical gardens on the novitiate premises (of course, with the help of the novices). Emile Gombert (1866–1948) collected orchids and established a garden dedicated to orchids (which survives till date). Louis Anglade (1873–1953) documented local plants through a collection of nearly 2000 paintings. George Foreau (1889–1959) assembled a collection of mosses, lichens, algae, and fungi while Alfred Rapinat (1892–1959) collected flowering plants and ferns. These Jesuits and the young novices often sent plant specimens to the Royal Botanical Gardens at Kew for proper identification. Santapau, though not a resident of Shembaganur, had begun his life’s work at the Kew Garden. Soon enough, the French Jesuits encouraged the young novices to interact with Santapau, who was then in the Jesuit college of St. Xavier’s at Bombay (1940). It was only expected that the younger Jesuits would follow his botanical legacy.

Jesuits with the oldest tree on the Palni Hills 1903 - Courtesy Rapinat Herbarium Trichy

Jesuits with the oldest tree on the Palni Hills, 1903. Courtesy Rapinat Herbarium Trichy.

As a young novice at the Sacred Heart College, KM Matthew (1930-2004) was acquainted with botanical surveys in Shembaganur. With the nomination of Santapau to the Botanical Survey of India, Mathew was encouraged to pursue his doctoral research with the senior botanist. In 1962, under the supervision of Santapau, KM Matthew became the first Indian Jesuit to acquire a PhD in botany, focusing on the alien plants of the Palni Hills. In 1963, another pupil of Santapau, Cecil Saldanha (1926-2002) was awarded his PhD for his thesis on Taxonomic Revision of the Scrophulariaceae of Western Peninsular India. Like Santapau, both the Jesuits made significant contributions to plant taxonomy: KM Mathew published the four-volume Flora of Tamil Nadu Carnatic (1981-1988) while Saldanha published Flora of the Hassan District, Karnataka (1976).

Matthew and Saldanha were among the first Indian Jesuits to engage with science in a globalized, industrial era, where science and technology came to be seen as intertwined with “social, political and cultural issues of societal relevance”. Observing the wider implications of modern science and technology for the Catholic Church, its bishops observed at the Second Vatican Council (1962-65) that “if these instruments (of science and technology) are rightly used they bring solid nourishment to the human race”. After Vatican II, the Jesuit Superior General of the Jesuits, Pedro Arrupe (1907-1991) named the first delegate for what came to be known as the “scientific apostolate” of the Jesuits. In 1979, the Jesuit scientists of India, Nepal and Sri Lanka came together to organize the first ever meeting of south Asian Jesuit scientists. Responding to a report of this meeting, Arrupe noted the “apostolic aspect of the Jesuit [s]cientist’s work” and emphasized the need for Indian Jesuits “to reflect more on Indian problems”. This provides a hint of how the “new” Jesuit scientific activity became “localized” in the backdrop of the Catholic Church’s wider embrace of modern science and technology. Further investigation of how Jesuit science is manifested locally in the context of a global missionary ethos is required, especially with respect to Jesuit botany in southern India.

Contemporary Jesuit botanists in India have widened their horizons beyond the plant taxonomy of their French mentors. This engagement has extended into specialized terrains like molecular systematics and agricultural biotechnology for solving “Indian problems” like drought, crop pests and diseases, extinction of native flora, and deforestation. While Jesuits have also ventured into the twenty-first century (and contentious) disciplines like genetic engineering, there is little scholarship on how the trajectory of Jesuit biological sciences evolved in India. The establishment of the Shembaganur novitiate and the arrival of European Jesuits like Santapau signify a milestone for exploring the dawn of post-Restoration Jesuit science in southern India.

The election of the first Jesuit Pope (Francis) in 2013 has renewed interest in Jesuit studies. Historians of Jesuit science and historians of post-colonial science could consider stepping into this uncharted domain of why the quintessential Jesuit botanist did what he did in independent India. Young historians of Jesuit science must wonder why then Prime Minister of India, Indira Gandhi observed after Santapau’s death in 1970 that:

In Rev. Fr. Santapau’s death we have lost an eminent scholar who has served education and science for over 40 years. His deep love for India urged him to become a citizen of the country. He had a great knowledge of, and concern for, our plant wealth and wrote intensively on it for experts and laymen. May his memory long continue to inspire all those interested in our flora.

Joseph Satish V is a PhD student in Science, Technology and Society Studies (STS) at the Centre for Knowledge, Culture and Innovation Studies (CKCIS), University of Hyderabad, India. His research focuses on the work of Jesuit scientists in the botanical sciences in independent India.