The World of Structural Biology Dylan Heussman December 14, 20202 Meet “Anton”

Anton is a massively parallel supercomputer designed and built by D. E. Shaw Research in New York, first running in 2008 Super computers for biology research?

• Anton is a special-purpose system for molecular dynamics (MD) simulations of proteins and other biological macromolecules. • Named after Anton van Leeuwenhoek who is often referred to as "the father of microscopy." • People all around the world are using extremely fast and expensive computers to simulate biological structures, WHY?

The original “Anton” What is Molecular Dynamics?

• Molecular dynamics is a way of computationally pushing atoms around based on instructions informed by physical laws. • The computer digitally calculates Newton’s equations of motion and finds “trajectories” based on initial conditions. Molecular Dynamics simulation Water can often times be very interesting of water in an “MD” box. itself, but we can use molecular dynamics to tackle much larger problems! Molecular Dynamics and Biomolecules

• Computer simulation is an invaluable tool for studying biomolecular structure and dynamics, the relationship between which is central to understanding important cellular processes. • Proteins have many moving parts (many degrees of freedom), which requires a lot of computational power to simulate. • Proteins interact directly with the aqueous environment – you can’t leave out the water and the ions, and expect the proteins to behave! “Ribbon” structure of a ATP binding cassete transporter protein protein in a simulation– here they’ve “coarse- grained” from the atomic view to the secondary structure just to simplify things. Notice the hydrophobic interaction with lipids! Structural Biology 101

• A protein’s structure allows it to perform its job! If we don’t understand protein structure, we can’t know what it does. • The structures of nucleic acids are also critical to their function in gene expression and replication (remember the central dogma?). • Immune-system proteins bind to foreign molecules such as bacteria and viruses with one end while recruiting other immune-system proteins with the other. • Understanding how biomolecules interact and bind to their targets helps us to develop drugs to treat diseases, and to understand how incorrect structures interfere with normal function. Protein Basics • What is a protein? Proteins come in thousands of different varieties, but they all have a lot in common. For instance, they're made of the same stuff: every protein consists of a long chain of joined-together amino acids. • What are amino acids? Amino acids are small molecules made up of atoms of carbon, oxygen, nitrogen, sulfur, and hydrogen. To make a protein, the amino acids are joined in an unbranched chain. • If we know the structures of amino acids and how they are connected in a chain, do we know what shape a protein folds into? NO! Even though a protein’s amino acid sequence might be known, it doesn’t tell us about the folded structure directly. Proteins are incredibly diverse in their function!

Hemoglobin Ligand-gated ion channel

Collagen How Do Proteins Fold? Recent Breakthroughs on Protein Folding (Google’s Deep Mind) • The protein folding problem is regarded as one of the grandest biochemistry challenges of the last 50 years • AlphaFold ai uses artificial intelligence to deliver relatively quick answers to questions about protein structure and function that would take many months or years to solve You Can Help Protein Structure Scientists! • Protein structure prediction: Knowing the structure of a protein is key to understanding how it works and to targeting it with drugs. The number of different ways even a small protein can fold is astronomical because there are so many degrees of freedom. • Foldit attempts to predict the structure of a protein by taking advantage of humans' puzzle-solving intuitions and having people play competitively to fold the best proteins. • Protein design: Since proteins are part of so many diseases, they can also be part of the cure. Players can design brand new proteins that could help prevent or treat important diseases. • FoldIt even includes proteins associated with coronavirus infection! Foldit Playthru:

• Proteins want to adopt structures that are thermodynamically Protein secondary structures stable– they tend to form alpha helices and beta sheets. stabilized by hydrogen bonding • Foldit allow you to manipulate proteins in space and penalizes between nearby amino acid folded structures which bring amino acid structures too close to one residues. another. Can you guess what these structures do?

DNA Origami Structure with HIV antigens attached “Nanobox” Engineered out of Fenna-Matthews-Olson DNA Origami! Complex Any questions? Now that we have seen how scientists tackle molecular structure using computers, let’s see how this is done in a laboratory! Rosalind Franklin and the Discovery of DNA Structure

1913 : X-ray diffraction: Max von Laue, William Henry Bragg, and William Lawrence Bragg discover that crystals diffract X-rays, producing an orderly pattern that can be used to deduce the location of every atom in the crystal.

1951: Studying DNA structure with X-ray diffraction, Franklin and her student Raymond Gosling took pictures of DNA and discovered that there were two forms of it, a dry "A" form and a wet "B" form.

1953: Watson and Crick’s solution was formally announced following its First X-Ray diffraction pattern of DNA publication in that month’s issue Rosalind Franklin of Nature magazine. X-Ray Crystallography

• X-rays from a synchrotron source are bombarded upon a crystallized biological sample (proteins, DNA, RNA). • When the X-rays interact with the atoms in the sample they diffract off in ways unique to that molecule. • Scientists then interpret the diffraction pattern to solve for the structure of the molecule which made it. Protein Data Bank: Where the structures go!

• Scientists are a highly collaborative group of people! • Muscle's myoglobin, the first protein structure revealed: After 22 years of work, John Kendrew of the Cavendish Laboratory in Cambridge (UK) uses X-ray diffraction to determine the 3-dimensional structure of myoglobin, the first protein to have its structure determined. • Structures now come into Protein Data Bank at an average rate of more than 10 per day. • The total number of accessible structures is greater than 24,000.

Protein structures from the protein data bank Cryo-electron Microscopy Macromolecular Complexes • In molecular biology, large complexes of different proteins interact to form larger functional units. • Proteins join to form multimers, protein-DNA and –RNA complexes. • How do we learn about structures that include multiple subunits?

Free trp operator DNA, and in a complex with the Trp repressor protein (Harteis IMJS, 2014) Gel Electrophoresis Experiments

ThermoFisher Native Mass Spectrometry High Resolution Confocal Microscopy

Confocal microscopy of dendritic cells (immunology.org) Using Spectroscopy to Calculate Molecular Structures • Fluorophores can be used to image A B particular cell areas/proteins but they can also be used to measure structure indirectly through spectroscopy. • In our group, we use cyanine molecules to tag DNA and are able to monitor how structural changes to the DNA change the absorption spectrum of the A) The absorption and emission spectrum of the cyanine dyes. Cy3 molecule. B) The molecular structure of the Cy3 and Cy5 fluorophores. By covalently inserting cyanine dyes into the sugar-phosphate backbone of DNA, the molecules report on the local structure of the DNA at the labeling position. Absorption (Optical Density) Circular Dichroism

• Circular dichroism is especially useful for measuring the optical properties of chiral entities. • Chirality is a special form of asymmetry which is not present in Cy3 alone, but emerges when Cy3 is forced to interact with another Cy3 within the structure of DNA– this is because DNA itself is chiral! When two cyanine molecules are stuck in close proximity to one another in DNA, their optical properties change due to coupling between them.

The measured spectra of the molecules changes when the structure of the DNA region they are in changes. As the angle between the dyes changes, the resultant spectra also changes.

By measuring the absorption and circular dichroism spectrum of the molecules we can assign a structure which would generate the observed spectrum. Why do you think it would be useful to identify and characterize the structures at different DNA junctions?

Takeaways

• Structural biology represents an extremely diverse set of researchers taking many different approaches to the problem. • Most major universities have labs which use nearly all of the techniques we touched upon today. • The study of biomolecule structure is extremely important to the maintenance of human health. • Its never too early to get involved! Thank you for listening! Any questions?

Quantum Computer

• The problem of simulating large proteins….