Before we model insulin, it is good to understand the BIG picture of what insulin is, and why it is important to our health. Watch this videos below for a good overview, as well as the nice review entitled "Insulin and its receptor: structure, function, and evolution".

"The Insulin Receptor: a Prototype for Dimeric, Allosteric Membrane Receptors?" by Pierre De Meyts

It's amazing to see how our understanding of insulin has steadily progressed over the past century and a half! If you follow this timeline, you can see how knowledge of insulin and diabetes developed slowly over time and how each discovery was built upon our understanding from previous discoveries. This is the "process of science"!

1869 Paul Langerhans discovers collection of pancreatic cells "Islets of Langerhans".

1889 Oscar Minkowski and Joseph von Mering remove a dog's pancreas and discover flies attracted to the sugar in the dog's urine.

1901 Eugene Opie showed that destruction of the Islets of Langerhans results in diabetes.

1921 Frederick Banting and Charles Best discover insulin in the laboratory of John Macleod. Bertram Collip was a biochemist that purified their extract for human injection. This video is a re-enactment of the discovery of insulin. It's old, long, and a little hokey but it does a great job of putting you back in the time of when the mechanism of diabetes was unknown and understanding the experiment done to discover it.

1922 The first medical administration of insulin is given to a 14 year old diabetic boy using insulin purified by Collip. The boy lives for another 13 years before dying of pneumonia.

1923 Nobel Prize in Physiology or Medicine: Banting and Macleod received the award, who later shared it with Best and Collip. The award to Banting and Macleod caused some controversy. If you watch the video "The Quest" above, why do you think that might have been the case?

1936 Danish physician Hans Christian Hagedorn discovered that the activity of insulin can be prolonged with the addition of protamine.

1950 Neutral protamine Hagedorn insulin (NPH) is marketed as an intermediate acting insulin by Danish pharmaceutical company Novo Nordisk.

1955 Sanger determines the sequence of insulin, the first protein to be fully sequenced. This video interview with Sanger is long but if you advance to 22:00, he begins talking about his research on insulin. He mentions that insulin was one of the few proteins available in pure form at this time, which allowed him to determine its sequence.

1958 Nobel Prize in Chemistry: Frederick Sanger for the "structure" of insulin. Really, he published the sequence of insulin or the primary structure.

1963 Insulin is first human protein to be chemically synthesized.

1964 Nobel Prize in Chemistry: Dorothy Hodgkin for structure of penicillin, Vit B12. She becomes the 3rd woman to receive the Chemistry Nobel and only the 4th ever! Only Ada Yonath has been so honored since then - in 2009 for the structure/function of the ribosome.

1969 Hodgkin discovers structure of insulin - 5 years after her Nobel Prize! At the end of this video is a clip from an interview she did near the end of her life, reflecting on her discovery of the insulin structure and the importance of Sanger's previous contribution of the sequence. See the attached heartfelt reflection on Dorothy's life by Max Perutz (our hemoglobin guy from last year!) entitled "A Passion for Crystals".

1978 Genentech uses recombinant DNA techniques to produce synthetic "human" insulin.

1982 Novo Nordisk introduces Insulin Pen.

1992 Medtronic releases insulin pump.

1996 Eli Lilly markets first insulin analog, lispro.

2000 Islet cell transplantation for DM1 patients.

2013 Artificial pancreas developed by Univ of Cambridge - insulin pump + glucose monitor.

2015 Edward Damiano develops iLet, bionic pancreas that delivers insulin or glucagon every 5 min as required.

We are going to start out by modeling the basic structure of an insulin molecule. As you will see, it has a very unique structure. In order to do this, you will need access to the 3DMD mRNA to Protein Insulin Folding Kit (available for purchase or loan), some toobers and cysteine sidechains, or other materials that you might think of (pipe cleaners, etc).

If you do not have access to the kit, you will need toobers or other materials to model the two peptide backbones: A chain = 21 in; B chain = 30 in. You will need to add cysteine sidechains, alpha helices and beta sheets according to the map below.

Now you need to fold the 2 chains in the proper structure. The A chain will look like an upside down "U" with a flat section between the 2 helices. The B chain looks like a "Z" with the alpha helix being the diagonal section. The A chain will have one intrachain disulfide bond between A6 and A11. Then there will be 2 interchain disulfide bonds; one between A6 and B6 and one between A20 and B19.

Use the handout below to help you explore and model insulin. Take your time, ask lots of questions, discuss, and have fun. I'll summarize later in a video.

Thought question: What "level" of protein structure did you just model? Primary, secondary, tertiary, quaternary? Discuss!

Modeling Insulin Transcription and Translation

So now that you have a good idea of the structure of insulin, you might be asking yourself a lot of questions. For example, why 2 chains? Why so many disulfide bonds? How do the the correct cysteines find each other?

First, make sure that your team understanding the "Central Dogma of Molecular Biology". That's just a stuffy way of saying that DNA is the code for messenger RNA (or mRNA) which is the code for a protein. If your teacher has the 3DMD Flow if Genetic information Kit, you can review these processes with that. Also, take a look at the RNA coding activity handout below. You can use either the attached codon chart or circle (your choice) to "transcribe" DNA to RNA and "translate" RNA to protein. In other words, you will "crack the code" of the insulin gene!

Modeling Insulin Synthesis and Processing

Now that you understand how the DNA within the insulin gene encodes the RNA and the RNA encodes the sequence for the insulin protein, it is time to model how insulin is synthesized and processed. The mRNA map below show the fully transcribed mRNA and the 3 possible reading frames for protein translation. As you go through the handout below using the mRNA map as a reference, you might want to model insulin processing with different colored pipe cleaners twisted together.

But first, let's make sure you familiar with the Golgi so that you can understand its role in insulin processing. I'm going to steal one of Science with Tom's lyrics for MAPS:

"The cell does a lot, so we're gonna model it, model it, model model like it's hot!

Insulin Storage and Secretion

After modeling insulin synthesis and processing, you should now understand that the insulin translation product is called preproinsulin. This includes the A and the B chain that we modeled earlier and 2 peptide segments that are not found in the final product, the signal peptide and the C peptide. This precursor protein needs to be processed into the final product which is then secreted by the pancreatic beta cells.

The modeling activity you just did gave you a simplistic idea of how that might happen. Turns out it is a little more complicated than that. I found this figure in the review I've attached below. You can look at the review if you are interested but I thought the figure was cool. Take a close look at this figure and make some observations. Does anything surprise you about this figure? Without reading the paper, can you use the knowledge of insulin synthesis that you have constructed so far (along with anything you might remember from Biology class ;)) to make some statements that describe what is being depicted here? Don't worry about understanding all of it or being "correct". There is a lot of detail here. Just see what looks familiar and try to fit it in to what you think you know. Discuss as a team how you think the system might work based on this figure. We'll discuss in the upcoming summary video.

When I was working with the Rat Genome Database (rgd.mcw.edu), a worldwide genomics resource for researchers who use rats as disease models, I worked with a software developer to create a pathway program that allows exploration of the cellular signaling pathways involved in these actions and links to the important genes. Feel free to play around with it. If you hover over the dots on the arrows, text will appear to explain the pathway. You can also select "Show Callouts" on the toolbar. If you click on the specific organs/tissues , it will take you to a pathway for that. There are also common intracellular signaling pathways (labeled with a big C) that are involved that you can click on to see the specifics of those. You can click on proteins to see their gene record. There are lots of hidden tricks - just click away! Unfortunately, we never finished the project before I left but it still is interesting to explore and learn from.

Click on this link to get to the RGD Physiological Pathways for insulin:

Insulin receptor and how insulin binds to it is another interesting topic. If this is something you would like to model, please see some of the reviews as well as structure papers attached below.

Insulin Engineering

An important area of insulin research today is protein engineering to build a better insulin to treat patients with Diabetes Mellitus Type 1 and 2. There is a lot of effort being made to make small changes to our native insulin protein to allow it to have slightly different properties related to activity and stability while not interfering with its ability to bind to its receptor. I am working with MSOE Biomolecular Engineering students this year and they are working on a project to model various analogs to help show the rational design being used to develop these new drugs. Modeling one of these analogs would be an awesome final project for a MAPS Team as well. See the papers attached here if you are interested in pursuing this for your protein story

Defining Your Insulin Story

Your MAPS Team will want to have a clear idea of what your insulin story is before you start your model design. And one of the best ways to define your protein story is to write a scientific abstract.

Abstracts are, by definition, short. You might want to start with one sentence for each of the sections listed below, and then add additional sentences as needed.

• Introduction: What is the overall relevance of your protein of interest?
• Question: What is the particular story of your protein that your team will model?
• Findings: What answers to your question were you able to determine through your modeling project? Be specific as to what structural elements your team has modeled and how they are relevant to your story.
• Conclusion: How do your findings relate to the big picture? What important questions related to your protein story are still being actively researched?

Click here to see a sample abstract

Once you have defined the insulin protein story you want to tell, you will need to find a protein structure file that you can work with in Jmol to design your 3D printed insulin model.

Designing and building your own physical insulin model will give you great insight into specific structures that are important to the insulin protein story you have chosen to focus on. Even if you don't have your design 3D printed, studying the 3D design in specialized computer software is still helpful.

Below are a few additional links that may help you in your insulin structure search.

• Jmol Training Guide - Finding Protein Structures
• RCSB Protein Data Bank
• Insulin Molecule of the Month
• Designer Insulins Molecule of the Month
• Insulin Receptor Molecule of the Month
• Insulin and Diabetes Poster from the PDB101

"Designing" a protein model means you explore a protein structure in Jmol, and then simplify the way the protein is visually displayed to make the key features of the protein that help communicate your molecular story more obvious.

This can mean hiding some atoms that are not important for your protein story, changing the display format of certain parts of your protein structure or changing colors to best highlight the most important parts of the protein structures. In the next section, you will learn the Jmol commands needed to accomplish this.

All 3D printed protein models made in the MAPS program are designed using the program Jmol. You start with a protein structure file (.PDB file), which contains the 3D locations of all of the atoms that make up the protein. Using the Jmol commands you will learn below, you can then edit the way the protein is displayed, hiding some atoms, changing display formats and customizing colors. When happy with your design, you can export your protein model from Jmol in file formats suitable for 3D printing.

We have created a detailed Jmol Training Guide that will cover everything you need to know to design and build your model. The Jmol Training Guide is broken down into four main sections, which we strongly recommend you explore in order until you are comfortable with Jmol and designing protein models for 3D printing.

• 1) Getting Started:
     - Accessing Jmol
     - Loading Structures
     - Saving and Reloading
• 2) Simplifying Your Protein:
     - Exploring the Structure
     - Focusing on the Backbone
• 3) Adding the Details:
     - Adding Sidechains
     - Adding Ligands
     - Adding Molecular Bonds
• 4) 3D Printing Your Design:
     - Adding Support Struts
     - Checking Your Design
     - Print it Yourself
     - Pay Someone Else to Print it