When I came across a flyer for the above, கொஞ்சம் ஆர்வக் கோளாறுலேயும் கொஞ்சம் அசட்டு தைரியத்துலேயும், registered immediately.
- ஆர்வக் கோளாறு - when else will i get a chance to attend a Nobel Laureate lecture in person?
- அசட்டு தைரியம் - the speaker is bound to know the average knowledge base of the audience. After all it's a public forum.
Came the day, I was happy to be accompanied by Gopu! (who I was sure would understand more than me & may not be averse to a few explanations.)
Let me confess - I left Chemistry and all other Science subjects behind in High School, all of 40+ years back. Am not very updated on current happenings in the Practical Scientific world, leave alone Research! In fact I was happy, Dr. Kobilka is an American and I could at least pronounce his name properly and would understand his accent. (The few times in my life, when my Hollywood addiction is put to good use.)
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| IIT and Vallabha |
The Talk....
At the outset, it was amusing to hear Dr. Kobilka say, 'This was his largest audience and he was nervous!' If most of the audience was like me - he had no idea, how nervous we were.
But surprisingly he started on a non scientific note. Highlighting his background, his academic journey, the various institutes he had associated with etc. This put the audience, at least me - at ease. I was all ears & comprehending. Slowly Science crept in, but I was still in sync - Thanx to the simple Graphic slides!
Dr. Kobilka's Early life....
(Growing Up In Little Falls, Minnesota)
Born into a baker's family in Little Falls, Dr. Kobilka said the town was hardly 8sq miles in area (No more than 3 miles in any direction) with a max population of 7500. The 3D map showed the relative locations of various buildings in the town and his first job was slicing bread in his father's bakery. It was a family owned business run by his grandfather.
He attended St. Mary’s elementary school through the eighth grade then moved onto Little Falls High School. His interest in science started with wanting to become a physician. Alas! there was no usual noble thoughts behind this, but the mere impression that local physicians were given a lot of respect.
His favorite classes in high school were math, physics, chemistry and biology. (Fortunately I had Maths in common with him!)
Undergraduate Years....
After school, he entered the University of Minnesota, Duluth to prepare for medical school, in 1973.
In his very first term, he was lucky enough to meet both his future wife (Dr. Tong Sun Thian) and his mentor Professor Conrad Firling. He met Dr. Tong the very first week, in the Biology Lab, and they seem to have been together since.
Professor Conrad Firling, was a biology professor, who was willing to take undergraduates into his lab to work on projects in developmental biology. Under him Dr. Kobilka learnt
- first to wash glassware, in a lab, properly.
- later to develop an organ culture medium for studying the salivary glands of some insects and their chromosomes.
(Science was beginning to creep in & I've no idea what those insects were!)
He also worked on a summer project with Professor Robert Carlsen, an organic chemist and this sowed the seed for a growing interest in basic research.
Majoring in Biology and Chemistry, he applied to ten medical schools, but was selected only in 2. Fortunately for him, one of them was Yale and he moved there from Duluth.
Yale University Medical School....
At Yale, as part of their curriculum, all medical students were required to write a thesis based on original research.
Dr. Kobilka's very first research project involved dengue fever, on which he worked, through a summer in a lab in Malaysia, involving both field and bench research.
For his thesis project, he worked with Professor Denis Knudsen, a virologist, studying the genetic diversity of rotavirus, a common cause of gastroenteritis in children.
According to him, both these projects were not successful, because they were short termed and gave no fresh scientific insights, into their respective domains. Their only benefit was reinforcing the passion for pure research in him.
After his first year of Med School, he married Tong Sun, who was not a Doctor yet.
Clinical Training At Barnes Hospital...
Owing to financial constraints, a career in research was not an immediate option and Dr. Kobilka decided on a residency in internal medicine at Barnes Hospital, affiliated with Washington University School of Medicine in St. Louis, Missouri, for 3 years.
Dr. Kobilka explained that The Human nervous system can be divided into two functional parts: the somatic nervous system and the autonomic nervous system. The autonomic nervous system regulates many of the internal organs through a balance of two aspects, or divisions. The two divisions of the autonomic nervous system are the sympathetic division and the parasympathetic division. The sympathetic system is associated with the fight-or-flight response, and parasympathetic activity is referred to by the epithet of rest and digest. Homeostasis is the balance between the two systems. At each target effector, dual innervation determines activity. For example, the heart receives connections from both the sympathetic and parasympathetic divisions. One causes heart rate to increase, whereas the other causes heart rate to decrease.
During his training at Barnes, his interest in intensive care medicine, led him to continuously take rotations at the 3 intensive care units - medical, pulmonary and cardiac. Here he found that, patients were typically unstable and required urgent medication
- to regulate their heart rate and blood pressure
- to control pain.
He went onto mention that these medications acted on GPCRs (G protein coupled receptors), which are of several types.
Those regulating BP and pulse are called adrenergic and muscarinic receptors, while those controlling pain are opioid receptors.
By now, I was at sea and slowly drowning. The talk was going above my head. I, ofcourse, went back and googled and somewhat surfaced from deep water. Sharing some of that here....
What are GPCRs?
GPCRs are proteins located in the cell membrane that binds extracellular substances and transmits signals from these substances to an intracellular molecule called a G protein (guanine nucleotide-binding protein).
GPCRs are found in the cell membranes of a wide range of organisms, including mammals, plants, microorganisms, and invertebrates. There are numerous different types of GPCRs—some 1,000 types are encoded by the human genome alone—and as a group they respond to a diverse range of substances, including light, hormones, amines, neurotransmitters, and lipids.
Nearly every function of the human body, from sight and smell to heart rate and neuronal communication, depends on G-protein-coupled receptors (GPCRs). Lodged in the fatty membranes that surround cells, they detect hormones, odours, chemical neurotransmitters and other signals outside the cell, and then convey their messages to the interior by activating one of several types of G protein. The G protein, in turn, triggers a plethora of other events. The receptors make up one of the largest families of human proteins and are the targets of one-third to one-half of drugs. Working out their atomic structure will help researchers to understand how this central cellular-communication system works, and could help drug-makers to design more effective treatments.
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| The Net |
Beginning at the N-terminus, this long protein winds up and down through the cell membrane, with the long middle segment traversing the membrane seven times in a serpentine pattern. The last of the seven domains is connected to the C-terminus.
When a GPCR binds a ligand (a molecule that possesses an affinity for the receptor), the ligand triggers a conformational change in the seven-transmembrane region of the receptor. This activates the C-terminus, which then recruits a substance that in turn activates the G protein associated with the GPCR. Activation of the G protein initiates a series of intracellular reactions that end ultimately in the generation of some effect, such as increased heart rate in response to epinephrine or changes in vision in response to dim light.
The existence of GPCRs was demonstrated in the 1970s by American physician and molecular biologist Robert J. Lefkowitz. Lefkowitz shared the 2012 Nobel Prize for Chemistry with his colleague Brian K. Kobilka, who helped to elucidate GPCR structure and function.
In 1984, after his residency, Kobilka applied for cardiology fellowships, and he was particularly interested in the program with Robert Lefkowitz at Duke University in Durham, North Carolina. It was the premier lab studying receptors for adrenaline, which had become a model system for all hormone receptors. This gave him the opportunity to explore basic research in an area relevant to cardiovascular and intensive care medicine.
It is interesting to note that Dr. Kobilka, never became a cardiologist.
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| With his young family started in St. Louis. |
Fellowship Training At Duke University...
Joining the Lefkowitz lab, Dr. Kobilika found that, almost everybody there, seemed to know more than him and he was yet to familiarise himself with the research work being done there and the techniques they were using.
When Kobilka joined, the lab was just starting to think about how to clone the gene for the β2 adrenergic receptor (β2AR) and determine its genetic sequence. But the receptor was produced in such small amounts that the team was only able to collect enough protein to work out a few scraps of its likely genetic sequence.
From now on, we were in the midst of Scientific talk about Scientific research, and there was a lot, that I did not understand or misunderstood. I tried correcting a lot of my misunderstandings with further reading, but there may still be errors here - a direct result of my limited understanding.
Kobilka, joining the programme, decided to construct a library of mammalian genomic sequences and screen it with the scraps of sequences they had. This would pull out longer clones that could be pieced together to reveal the full sequence. However, when the team stitched together the receptor sequence, it had a real Eureka moment: several strings of amino acids that are typically found in cell membranes showed that the receptor snaked through the membrane seven times, just like rhodopsin, the light-detecting receptor in the retina that was also known to activate a G protein.
It was a surprise that, these receptors looked alike, one turned on by light, and the other by a hormone.
At the time, about 30 proteins were known to turn on G proteins, and they concluded that it was a whole family of look alike receptors. This family became known as seven-transmembrane receptors, or GPCRs, and is now known to have nearly 800 members in humans.
Dr. Kobilika, then, wanted to understand receptor structure and how the receptor worked in molecular detail. Thus the project extended to obtaining a crystal structure. Several years earlier Deisenhofer and Huber had obtained the first crystal structure of a membrane protein, proving that membrane proteins could be crystallized and demonstrating the value of protein structure in understanding mechanisms. However, the photosynthetic reaction center was a naturally abundant protein that could be obtained from bacteria. In contrast, even in lung tissue, where the β2AR was most abundant, it represented a very small fraction of membrane proteins.
Stanford University...
Around this time (1989), at Stanford, Professor Richard (Dick) Tsien, had just moved from Yale, and was building a new Department of Molecular and Cellular Physiology in the Beckman Center. Dr. Kobilka was offered a junior faculty position, which he accepted and moved to Stanford. To make ends meet, he moonlighted as a doctor in the emergency department at weekends.
In the lab they focused on two objectives:
- understanding the structure and mechanism of activation of the β2AR and
- determining the physiologic role of specific adrenergic receptor subtypes.
Cloning and pharmacological studies had identified 9 adrenergic receptor subtypes coded by 9 different genes: three βARs, three α1ARs, and three α2ARs. The drugs available at that time were not sufficiently selective to allow assignment of specific functions to each receptor subtype.
Dr. Kobilka wanted to see what the receptor looked like in three dimensions using X-ray crystallography, in which a beam of X-rays is fired at a protein crystal and the resultant diffraction pattern is used to reveal the arrangement of its atoms. However to produce an intelligible X-ray diffraction pattern, one first needed to crystallize the receptor- a formidable process of packing millions of identical copies of protein so tightly that they form a solid that looks like a microscopic shard of glass.
Working out the conditions that will allow a protein to crystallize can take years, and membrane proteins such as GPCRs are the hardest of all: they must be coaxed out of the membrane intact, but it is the membrane that holds them in shape. GPCRs are also constantly shifting into various states, and most are expressed in very low quantities. To collect enough β2AR protein, one had to express about 100-1,000 times the levels at which it is normally produced in a cell. Later, they also used fluorescence spectroscopy, one of the most sensitive biophysical techniques, to investigate receptor structure.
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| Gopu & Rajagopal V |
As the years rolled by, Kobilka's lab was carrying out various biochemistry and biophysics experiments aimed at getting to know the β2AR more intimately, and he was inching forwards in expressing and purifying the protein. But there were a lot of setbacks. They found that the GPCRs have big, floppy loops inside and outside and the receptor writhes and squirms, adopting a variety of levels of activation, which only made crystallization more and more difficult.
Finally, in late 2004, the group managed to grow tiny crystals, too small to be analysed at Stanford's synchrotron facility. Based on the suggestions of Gebhard Schertler, a crystallographer, Kobilka took his samples to the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, which had the tightly focused beamline needed to analyse such small crystals.
But the crystals diffracted to a resolution of only around 20 angstroms - so low that there was no discernible image. A resolution of about 4 A is needed to see the organization of individual atoms.
Further frustration followed, when the team couldn't get the crystals to grow any bigger or diffract any better. The receptor's changeable activation states and floppy segments, made it very difficult to trap all the proteins in an identical conformation. The team realized that they'd have to do something radical: chop off the loose ends, and either anchor the loop in place with an antibody or replace it altogether with a protein known to crystallize well.
The two approaches....
In 2005 Dan Rosenbaum and Søren Rasmussen, two very talented and intrepid postdoctoral fellows, joined the lab with the goal of crystallizing the β2AR. Søren and Dan took two different approaches to generate better quality crystals of the β2AR.
Søren identified antibodies that bound to a particularly flexible region of the receptor and Dan used protein engineering to replace the same region of the β2AR with T4 lysozyme (T4L), a highly crystallizable soluble protein. During 2006 crystals were obtained using both approaches combined with a newly developed lipid-based media known as bicelles consisting of a mixture of lipid and detergent. Initial crystals of the β2AR-Fab (3.4A) and the β2AR-T4L (2.8A) fusion protein complex, both diffracted to below 4A.
A trio of papers marked a milestone in structural biology, and intensified aggressive research into GPCR structures.
But these GPCR structures had been snapshots of receptors in an inactive state. To really understand the receptor's workings, researchers needed to see it as it was being activated by a ligand and turning on the G protein. This project was even more technically daunting than the last. The protein complex was too big to hold in the fatty scaffold; the G protein kept falling off; and this time, the extracellular part of the receptor wouldn't sit still for crystallization.
Dr. Kobilka reached out to all manner of experts for help and the various groups developed a detergent for stabilizing the receptor with its G protein; a lipid scaffold that could support the complex; and an antibody that could hold it together. And then they tested thousands upon thousands of crystallization conditions and ways to engineer the protein.
Finally, after another 5 years, they solved it and got a resolution of 3.2A revealing a tangled molecular threesome: β2AR with a ligand clasped at one end and the G protein nested up on the other.
The β2AR-Gs crystal structure was published in 2011 together with two companion studies using single particle electron microscopy and deuterium exchange mass spectrometry to characterize the dynamic aspects of this complex. These combined studies provided unprecedented insights into GPCR signaling at a molecular level.
It was for this pioneering work, that, The Nobel Prize in Chemistry 2012 was awarded jointly to Robert J. Lefkowitz and Brian K. Kobilka "for studies of G-protein-coupled receptors."
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| Brian. K. Kobilika receiving his Nobel Prize from H M King Carl XVI Gustaf of Sweden at the Stockholm Concert Hall on 10th Dec 2012. |
After this climax, there were 2 more sections of Epilogue...
Why 20 long years?
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| Gopu R |
According to Dr. Kobilka, this entire research took almost 20 years mainly because
- It was a new field and they almost started from scratch
- They had to learn how the receptors are built, how they transmit signals over the membrane and how they are regulated
- Purifying the Protein, Crystallizing it to a high level of resolution in both their active and inactive stages, ensuring their stability, even when drawn out of their shells - all took their own time.
- The mechanisms for diffraction were also not so developed and Computer Docking was in its developmental stage.
The entire team managed to sustain their interests, hopes and irrational optimism through these years because
- They constantly achieved incremental advances & successes that kept their hopes alive.
- They were simultaneously learning so many new things about the Human nervous system, especially with respect to the GPCR.
- The nurturing scientific & academic environment of which they were a part.
- Their family and friends and the entire scientific fraternity and last but not the least, his wife, who worked with him every step of the way, supporting him both professionally and personally.
- His own temperament, love for his work and the sheer joy he derived in research.
His post Nobel Work....
I had very poor understanding of this section and the Graphs shown.
This part of the lecture is paraphrased in my own words.
Along with a team Professor Brian Kobilka, discovered the drug called PZM21 after evaluating some three million different compounds. His research is mainly aimed at finding an alternate to Morphin as a painkiller.
Morphine, derived from the opium poppy, works by acting on a receptor in the brain that reduces pains, but it also affects a different receptor that can lead to fatal breathing problems in the event of an overdose. But PZM21, which so far has only been tested on mice, appears to act on the first receptor to about the same level as morphine without significantly changing the second one.
It also caused less constipation than opiate drugs, a factor that limits how much of the drug can be given. The new drug dulls the feeling of pain in the brain because it has a “potent, selective and efficacious” effect on the brain receptor involved in the sensation of pain, without dangerous side effects.
Studied against Morphine, on mice, PZM21 did not appear to affect their breathing and the painkilling effect of PZM21 lasted for up to three hours, “substantially longer” than the maximum dose of morphine. The “constipating effect” was also “substantially less than morphine” and the mice did not show signs of addiction.
Morphine addiction is a result of the activation of the brain’s dopamine reward circuits. When this happens in mice, they tend to run about a lot. But PZM21 had “no apparent effect on locomotion”.
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| Vallabha Srinivasan |
PZM21 is an experimental opioid analgesic drug that is being researched for the treatment of pain. It is claimed to be a functionally selective μ-opioid receptor agonist which produces μ-opioid receptor mediated G protein signaling, with potency and efficacy similar to morphine, but with less β-arrestin 2 recruitment.
In tests on mice, PZM21 was slightly less potent than morphine or TRV130 as an analgesic, but also had significantly reduced adverse effects, with less constipation than morphine, and very little respiratory depression, even at high doses.
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| Rajagopal V |
My own take aways from the lecture
- As a speaker Dr. Kobilka was not flashy, aggressive or imposing. Rather soft spoken, his talk was delivered like a classroom lecture in a low sonorous voice.
- Yet he was very impressive. Kept the audience fairly hooked.
- The 2 main aids to this were - His presentation slides, that were simple, clear and precise and
- His own unbridled passion for his work that came through even when he was describing complex and inanimate Protein structures.
Lastly, I must read up, atleast about Basics, before attending such lectures, if I want to optimise the experience.
Acknowledging Vallabha, Vidya, RV, Jayakamala & their kids -
என் சக ஆர்வலப் பயணிகள்!
A big Thank you to Gopu, who made this an enjoyable experience and matched my every step in weathering sudden winds, rains, pot hole filled roads and Chennai peak hour Traffic to attend this lecture. The upturned umbrella and his valiant attempt to turn it back remains etched in my mind!
It seemed like a metaphor.
Standing exposed in ur ignorance, as science rains all around u.
Thankfully, he straightened the umbrella soon!
