ConJ/MCB 544

CONJOINT 544 “Protein Structure, Modification Diversification and Regulation”


First five weeks of winter quarter, 2021 (Tuesday, January 5 thru Thursday, February 4)
        1.5 credits

(Note: This class is taught every year, with course coordination alternating between Barry Stoddard and Roland Strong.   Stoddard runs the class and teaches in odd years (i.e. 2021 etc).

Meeting Time:   Tuesdays and Thursdays 3:15 to 4:45 via Zoom.

Zoom Details:

                          Meeting ID: 955 4109 0334

                             Passcode: 679938



Barry Stoddard    FHCRC Basic Sciences/UW Biochemistry)  (Weeks 1 to 4)

Melody Campbell    FHCRC Basic Sciences/UW Biochemistry) (Week 4, part II)

Phil Bradley         FHCRC Public Health Sciences and Basic Sciences/UW Genome Sciences)  (Week 5)


Class grading requirements

1. Attend all sessions and participate in discussion and Q/A about readings and lectures.

2. Choose two of the classes ‘Problems to Consider’ from the ‘Weekly Terms, Concepts and Challenges’ document that accompanies the class syllabus to answer with original text and original figures and turn in both by the Friday of week 5 (February 5th).

3. Complete the Final Project (also found at the end of ‘Weekly Terms, Concepts and Challenges’ and turn in by Friday, March 12.


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 biological reactions.  Over the past 10 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 over the past 40+ years, and  especially  the recent development of powerful methods for protein 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, covering various aspects of protein structure and function such as:

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

(2) Protein folds and protein shape-shifting and moonlighting

(3) Protein modeling, fold design and structure-based stabilization and optimization

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

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

(6) Protein fold prediction and covariation analyses.

(7) Tandem Repeat Proteins

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.



Week One (Jan 5 / 7)                            

i.  Protein Structure and Folds: Definitions, Basics and Tools

ii. Protein sequence versus structure, protein stability


Structure Databases                                                        RCSB

Structure Illustration                                                        PYMOL

Structure Fold Classification and Analysis                           CATH             

Structure Identification and Homology Modeling                 PHYRE2         SWISSMODEL           I-TASSER

Structure Stabilization                                                      PROSS


1. Goodsell D. et al. and Burley SK. (2020) “RCSB Protein Data Bank: Enabling biomedical research and drug discovery” Protein Sci. 29 (1): 52-65.     doi: 10.1002/pro.3730        PMID: 31531901   

2. Burley SD et al. and Zhuravleva M. (2020) ”  CSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences” Nucleic Acids Research

PMID:  33211854      DOI: 10.1093/nar/gkaa1038

3. Dawson NL, Orengo C, Gáspári Z. (2020) “Assessing Protein Function Through Structural Similarities with CATH” Methods Mol Biol. 2112: 43-57.    doi: 10.1007/978-1-0716-0270-6_4         PMID: 32006277

4. Kuhlman B, et al. and  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

5. Lambert AR, et al. and Stoddard BL. (2020) “Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition” Structure 28 (7): 760-775.e8.

doi: 10.1016/j.str.2020.04.009            PMID: 32359399          


Week Two (Jan 12 / 14)                        

i. Structure-Function Studies via NMR

ii. Protein behaviors: shape-shifting, moonlighting, alternative splicing I


Structure Similarity Searches                                         DALI,   FATCAT


1. Tuinstra RL et al.  and Volkman BF. (2008) “Interconversion between two unrelated protein folds in the lymphotactin native state” Proc Natl Acad Sci 105 (13): 5057-62.    doi: 10.1073/pnas.0709518105

PMID: 18364395        PMCID: PMC2278211

2. 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

PMID: 14718922

REVIEW: Goodchild SC et al. (2011) “Structural gymnastics of multifunctional metamorphic proteins” Biophys Rev. 3 (3):143.    doi: 10.1007/s12551-011-0053-8      PMID: 28510063       PMCID: PMC5425669


Week Three (Jan 19 / 21)                     

i. Structure-Function Studies via Crystallography

ii. Protein behaviors: quaternary assemblages, cooperativity, allostery, alternative splicing


Electron Density and model visualization                       COOT   and MORE ON INSTALLING COOT


1. Jurica MS et al. and Stoddard BL. (1998) “The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate”  Structure 6 (2):195-210.        doi: 10p.1016/s0969-2126(98)00021-5         PMID: 9519410.

2. Dombrauckas JD Santarsiero BD and Mesecar AD. (2005) “Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis” Biochemistry 44 (27): 9417-29.       doi: 10.1021/bi0474923

PMID: 15996096

3. Christofk HR et al. Cantley LC. (2008) “The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth” Nature 452 (7184): 230-3.    doi: 10.1038/nature06734      PMID: 18337823

REVIEW:   Hsu M-C and Hung W-C (2013) “Pyruvate kinase M2 fuels multiple aspects of cancer cells: from cellular metabolism, transcriptional regulation to extracellular signaling” Molecular Cancer 17 (35).    

doi: 10.1186/s12943-018-0791-3           PMID:  29455645        PMCID:   PMC5817853


Week Four (Jan 26 / 28)                       

i.  Structure-Function Studies via CryoEM

ii. Protein behaviors: quaternary assemblages, DNA binding


1. Shen, B.W. et al. and Stoddard, B.L. (2021) “Assembly and coordination of a bifunctional restriction-modification enzyme bound to multiple DNA sites visualized by CryoEM” submitted.

2. Campbell, M.G et al. and  Nishimura, SL (2020) “Cryo-EM reveals integrin-mediated TGF-b activation without release from latent TGF-b“ Cell 180: 4980 -501.


Week Five (Feb 2 / 4)                

i. Structure-Function Studies via Computations      

ii. Tandem repeat proteins, scaffold and cages


1. Cao, L., et al. (2020). De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science, 370 (6515): 426–431.   PMID: 32907861    DOI: 10.1126/science.abd9909

2. Correnti, C. E. et al. (2020). Engineering and functionalization of large circular tandem repeat protein nanoparticles. Nature Structural & Molecular Biology, 27 (4): 342–350. PMID: 32203491

DOI: 10.1038/s41594-020-0397-5

3. Divine, R. et al. (2020). Designed proteins assemble antibodies into modular nanocages. In Cold Spring Harbor Laboratory (p. 2020.12.01.406611). (BioRxiV Preprint)