CONJOINT 544 “Protein Structure, Modification, Diversification, and Regulation”

First five weeks of winter quarter, 2025 (Tuesday, January 7 thru Thursday, February 6)
        1.5 credits

Meeting Time and Place:   Tuesdays and Thursdays 3:20 to 4:40 in the Fred Hutch Weintraub Building Room B1-072/074 (first floor, right side, past the Thomas Lobby Espresso Bar and Pelton Auditorium, across from Cafeteria and Director's Office)

Shuttle Schedule from UW School of Medicine:   https://www.fredhutch.org/en/about/contact-us/transportation/shuttle-schedule.html#fred-hutch-uw

Instructor(s):

Barry Stoddard    FHCRC Basic Sciences/UW Biochemistry)

Class grading

1. Attend  sessions and participate in discussion and Q/A about readings and lectures (vast majority of weighting)

2. Participate in any 'pop quizzes' focused on assigned readings (At instructor's discretion)

3. Complete an assigned Writing Project (to be described in Week 1) and turn in by Friday, March 7

Rationale and Background

While life likely arose from an RNA-centric origin, and many of the fundamental processes comprising the flow of genetic information and expression of biomolecules are governed by nucleic acids, proteins have evolved to carry out many of the core biochemical and biophysical processes required for life, such as generation of force and motion, creation of physical structures, transmission of material and information, and catalysis of most biological reactions.  Over the past 25 years, the development and use of proteins as therapeutic agents has increased dramatically, an advance that can be attributed to the enormous number of protein structures and mechanisms that have been elucidated, and  especially  the recent development of powerful methods for protein structural prediction and protein designa nd engineering

This course will provide a graduate-level survey of many of the fundamental properties of proteins that govern and define their folding, structures and function, and at the same time will introduce students to some of the most commonly used tools (and best practices) for modeling, analyzing and modifying protein structures and properties.  The class will provide detailed discussion and examples of how protein chemistry and structure/function analyses are employed, while also offering a survey of questions, approaches, results and 'greatest hits' from the past 25 years of protein-structure function analyses.

Over the course of the class, we will discuss all sorts of protein questions and topics, including:

(1) Examples of analyses of protein structure-function relationships via NMR, Crystallography, CryoEM and Computational Prediction and Design

(2) Basic principles governing protein folding and protein shape-shifting and moonlighting and the analysis of protein evolution, divergence and specialization via ancestral reconstructions

(3) Functional and structural consequences of protein alternative splicing.

(5) Functional and structural consequences of protein quaternary assemblage and multimerization, cooperativity and allostery.

(6) Protein design then and now: atomistic force-based design in 2003 and data-driven machine learning design in 2024

The course will assume knowledge at the level of an advanced undergraduate biochemistry course, and will be heavily skewed towards the use of structural information to understand the physical basis of protein behavior.  Emphasis will be placed upon the use and visualization of protein structural models as an essential component of fully appreciating protein behavior and function.  Background and understanding in the areas we will discuss at the level of Stryer, Biochemistry or Alberts, Molecular Biology of the Cell will be assumed.

SYLLABUS:

Week ONE     (Jan 7 / 9)       PROTEIN FOLDS AND PROTEIN ENGINEERING CIRCA 2003

Readings:

1. Anfinsen CB. (1973) Principles that govern the folding of protein chains. Science. 181 (4096): 223-30. doi: 10.1126/science.181.4096.223. PMID: 4124164.

2. Kuhlman B, Dantas G, Ireton GC, Varani G, Stoddard BL, Baker D. (2003) Design of a novel globular protein fold with atomic-level accuracy. Science 302 (5649):1364-8. doi: 10.1126/science.1089427. PMID: 14631033.

3. Watters AL, Deka P, Corrent C, Callender D, Varani G, Sosnick T, Baker D. (2007)N The highly cooperative folding of small naturally occurring proteins is likely the result of natural selection. Cell. 2007 128 (3): 613-24. doi: 10.1016/j.cell.2006.12.042. PMID: 17289578.

Extra Historical Reading(s)   (not assigned...just for your interest if you wish...)

The first 3-D structure of a protein (1958)

Kendrew, JC, Dintzis, HM, Parrish, RG, Wyckoff, H and Phillips, DC (1958) "A three dimensional model of the myoglobin molecule obtained by X-ray analysis" Nature 181: 662 - 666.   https://www.nature.com/articles/181662a0

de Chadarevian, S. (2018) "John Kendrew and Myoglobin: Protein structure determination in the 1950s" Protein Science 27 : 1136 - 1143.  https://onlinelibrary.wiley.com/doi/abs/10.1002/pro.3417

Websites and tools:

I want to make cool original figures of a protein model:  Pymol Download and Documentation: https://pymol.org    https://pymol.org/dokuwiki/

I want to find and download a structure or an AlphaFold Model:  Protein Database (RCSB):  https://www.rcsb.org

A DIY challenge for you: At the time it was designed, Top7 represented a unique protein fold, with no evolutionary history.  Now, almost 25 year later, a huge number of new protein sequences have been identified, and many, many new structures of those proteins have been determined or modeled. What is the most closely related folded protein to Top7 now? Does it still display a completely unique fold relative to those found in nature? Describe the similaritie and differences in the folded topology between Top7 and whatever naturally evolved protein domain you find that is similar to it.

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Week Two     (Jan 14 / 16)      PROTEIN evolution, DIVERGENCe, MOONLIGHTING and reconstruction

Readings:

1. Jeffery CJ. (1999) Moonlighting proteins. Trends Biochem Sci. 24 (1): 8-11. doi: 10.1016/s0968-0004(98)01335-8. PMID: 10087914.

2. Tuinstra RL, Peterson FC, Kutlesa S, Elgin ES, Kron MA, Volkman BF.  (2008) Interconversion between two unrelated protein folds in the lymphotactin native state. Proc Natl Acad Sci USA. 105 (13): 5057-62. doi: 10.1073/pnas.0709518105. Epub 2008 Mar 25. PMID: 18364395.

3. Chaloupkova, R., et al. And Damborsky, J. (2019) Light-emitting dehalogenases: reconstruction of multifunctional catalysts ACS Catalysis 9 (6): 4810 – 4823. https://doi.org/10.1021/acscatal.9b01031

Extra Historical Reading(s)    (not assigned...just for your interest if you wish...)

The first 3-D structure of an enzyme (1966)

Phillips, DC (1966) "The three-dimentional structure of an enzyme molecule" Scientific American 215.5:  78 - 93.  https://www.jstor.org/stable/pdf/24937166.pdf?refreqid=fastly-default%3Abd097da5ee00c4ba2b0f2ec47f938721&ab_segments=&initiator=recommender&acceptTC=1

Websites and tools:

I have a protein model (or a structure); what other structures does it resemble?   Dali Server:  http://ekhidna2.biocenter.helsinki.fi/dali/   FATCAT server: https://fatcat.godziklab.org/

I want to know more about moonlighting proteins:    MoonProt Database:  http://www.moonlightingproteins.org    

I'm working with an uncooperative protein; how do I fix it?   https://pross.weizmann.ac.il/step/pross-terms/

A DIY Challenge for you: One of the several moonlighting protein examples described this week was that of the aconitase enzyme from the Kreb's cycle, which moonlights as a DNA-binding iron-responsive transcription factor ('IRP').   The structure of the protein in its two functional states correspond to PDB ID code 1B0J or 2B3X (Aconitase) and 2IPY (IRP).  Download a PDB coordinate file for each version of the protein, and create a useful superposition of the two in PyMol that clearly illustrates the structural rearrangement corresponding to a change from one form to the other. Identify at least one residue that you believe to be under different structural/functional selection pressures for the two functions.

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Week Three    (Jan 21 / 23)       protein alternative splicing

Readings:  

1. Garcia J, Gerber SH, Sugita S, Südhof TC, Rizo J. (2004) A conformational switch in the Piccolo C2A domain regulated by alternative splicing. Nat Struct Mol Biol. 11 (1): 45-53. doi: 10.1038/nsmb707. Epub 2003 Dec 29. PMID: 14718922

2. Song Y, Zhang C, Omenn GS, O'Meara MJ, Welch JD. (2023) Predicting the Structural Impact of Human Alternative Splicing. bioRxiv [Preprint] doi: 10.1101/2023.12.21.572928. PMID: 38187531

3. Kane PM, Yamashiro CT, Wolczyk DF, Neff N, Goebl M, Stevens TH. (1990) Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science. 250 (4981): 651-7. doi: 10.1126/science.2146742. PMID: 2146742

Extra Historical Reading(s)    (not assigned...just for your interest if you wish...)

The first 3-D structure of an integral membrane protein (1985)

Deisenhofer, J. et al. and Michel H. (1985) "Structure of the protein subunits in teh photosynthetic reaction centre of Rhodopseudomonas viridis at 3Å resolution" Nature 318: 618 - 624.  https://www.nature.com/articles/318618a0

Websites and Tools:

I have a protein model (or structure) and want to identify and map conserved surface regions and residues:  CONSURF server:  https://consurf.tau.ac.il/consurf_index.php

A DIY Challenge for you:   Reading #2 above (Song et al., 2023) provides, in a supplementary table, a list of several high resolution crystal structures of 'reference' and alternatively spliced isoforms of the same protein, that they relied upon as a validating set of structures for their large-scale modeling exercise of alternative protein isoforms.  Identify and download any pair of related protein isoforms and analyze changes in packing and interactions within the protein fold that are created via the alternative splicing event.

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Week Four (Jan 28 / 30)           protein multimerization, cooperativity, and allostery

Readings:

1. Jurica MS, Mesecar A, Heath PJ, Shi W, Nowak T, Stoddard BL. (1998) The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6 (2):195-210. doi: 10.1016/s0969-2126(98)00021-5. PMID: 9519410

2. Reichard P. Ribonucleotide reductases: the evolution of allosteric regulation. (2002) Arch Biochem Biophys. 397 (2):149-55. doi: 10.1006/abbi.2001.2637. PMID: 11795865

3. Thomas WC, Brooks FP 3rd, Burnim AA, Bacik JP, Stubbe J, Kaelber JT, Chen JZ, Ando N. (2019) Convergent allostery in ribonucleotide reductase. Nat Commun. 10 (1): 2653. doi: 10.1038/s41467-019-10568-4. PMID: 31201319

Extra Historical Reading(s)    (not assigned...just for your interest if you wish...)

The first 3-D structure of a protein-DNA complex (1987)

Anderson JE, Ptashne M and Harrison SC (1987) "Structure of the repressor-operator complex of bacteriophage 434" Nature 326: 846-851.   https://www.nature.com/articles/326846a0

Websites and Tools:

I have a protein model (or structure) and want to examine molecular interactions and interfaces:  PISA server:  https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver

A DIY Challenge for you:  One of the two model systems presented this week, for the purpose of examining the inter-twined roles of protein multimerization, cooperativity and allostery, is pyruvate kinase. To generate a relatively complete picture, structures of three PK enzymes (from E. coli, Yeast, and Human) were shown at various points and compared.  Find and download at least two such structures, and compare their crystallographic packing and organization, from the standpoint of (1) the contents of the asymmetric unit in each, and (2) how the symmetry operators in each unit cell generates the biologically active enzyme tetramer.

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Week Five     (FEB 4  / FEB 6)        Protein structure prediction and design circa 2024

Readings:

1. Abramson J, et al., Hassabis D, Jumper JM. (2024) Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 630 (8016): 493-500. doi: 10.1038/s41586-024-07487-w. PMID: 38718835

2. Nomburg J, Doherty EE, Price N, Bellieny-Rabelo D, Zhu YK, Doudna JA. (2024) Birth of protein folds and functions in the virome. Nature. 633 (8030): 710-717. doi: 10.1038/s41586-024-07809-y. PMID: 39187718

3. Pillai A, Idris A, Philomin A, Weidle C, Skotheim R, Leung PJY, Broerman A, Demakis C, Borst AJ, Praetorius F, Baker D. (2024) De novo design of allosterically switchable protein assemblies. Nature 632 (8026): 911-920. doi: 10.1038/s41586-024-07813-2. PMID: 39143214

Extra Historical Reading(s)    (not assigned...just for your interest if you wish...)

The first 3-D structure of a protein that subsequently led to the development of a structure-based drug (1989)

Navia, M. et al. and Springer, JP (1989) "Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1" Nature 337: 615 - 620.   https://www.nature.com/articles/337615a0

Websites and Tools:

I want  to predict the structure of a newly identified protein-protein (or protein-DNA, or protein-RNA) complex:  AlphaFold3 server:  https://golgi.sandbox.google.com/about

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FINAL WRITING ASSIGNMENT (DUE FRIDAY, FEBRUARY 27)                       

Readings (If you wish...relevant to choice #2 of suggested writing assignments below):

de Crécy-Lagard V, et al. and , Xu J. (2022) A roadmap for the functional annotation of protein families: a community perspective. Database (Oxford) baac062.  doi: 10.1093/database/baac062. PMID: 35961013

Rocha JJ, Jayaram SA, Stevens TJ, Muschalik N, Shah RD, Emran S, RoblesC, Freeman M, Munro S. (2023) Functional unknomics: Systematic screening of conserved genes of unknown function. PLoS Biol. 21 (8): e3002222. doi: 10.1371/journal.pbio.3002222. PMID: 37552676

Your assignment (3 choices; for any of these strive for an interesting 2 page write-up plus figures):

Choice #1.  You be the instructor.  Choose a recently published study focused on an interesting and important aspect of a protein structure-function relationship and corresponding molecula rbehavior, and pitch it to me for inclusion in next year's rendition of ConJoint 544.   Provide a two page summary with at least one original figure generated by yourself using PyMol or an online analytic tool that explains who, what, when, and why the study rises to the level of being instruction-worthy, and how it fits into the theme of the class. The motivation can be that the underlying function of the protein is exceedingly important, or its molecular behavior is unique and fascinating (or even an exception to the usual rules of protein behavior), or the approach used is worth describing and working through.

Choice #2.  Tell me how this class has changed your thinking about your own research project.  Perhaps something we discussed and worked over in this class changed some aspect of how you're thinking about your own research project, or has given you a new approach (computational or experimental) to pursue as you work toward your thesis (or conduct your ongoing rotation). Tell me all about it...explain what the goal of your work in the lab has been, how you envisioned your approach originally,  and then what's new for you as you move forward.

Choice #3.  Build on existing insight and add knowledge.   If you go to the Protein Database (www.rcsb.org) or the AlphaFold Protein modeling database (https://alphafold.ebi.ac.uk) or the "Unknowme" database (https://unknome.mrc-lmb.cam.ac.uk) you will find many proteins that are annotated as 'unknown function' or 'domain of unknown function' for which there is very little in terms of biochemical or biological function. Beyond that, I can guarantee that the functional annotations provided for those that don't come up as 'unknown' are, in many cases, either flat-out wrong or are quite uninformative (i.e. 'kinase' or 'binding protein', etc).

Your assigment is to (1) read the really interesting final papers above, that describe this problem from the standpoint of a very broad community of molecular, cellular and structural biologists, and then (2) to find an interesting 'unknown function' protein from the Protein Structure Database or AlphaFold database or the Unknowme Database and to use all the tools, intuition, knowledge, and imagination you have at your disposal to provide a defendable (and hopefully testable) hypothesis regarding potential function.

How will you choose? So many interesting hits to consider. Personally, I'd go after something that flat-out looks really cool and complex.

How will you analyze?  Consider searching for all structural homologous proteins using DALI or FATCAT or the RCSB itself, as well as all sequence homologues, and see what pops up. Then, start looking for conservation patterns that might indicate functionally conserved sites. Look at stereochemical features of such areas of the proteins. Look at potential sites of covalent modification. Look to see if there are any publications linking the protein (and its gene) to a biological phenomena or phenotype.

How to impress me?  HAVE FUN,  write really clearly and cleanly, make original figures, and dig as deep as you can (OH...and keep it concise! This shouldn't go on for more than maybe about 1500 - 2000 words max)

AND AGAIN...if you've participated fully in the class to the point where you're now taking this on, you've already done just fine and have nothing to sweat regarding graduate graded credit for it. So, please don't turn this into a 'thing' other than seeing where webservers and your own thought processes take you.

THANKS FOR TAKING CONJOINT 544!

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