ConJ/MCB 544

Protein Structure, Modification, Diversification and Regulation

Course is Offered

First five weeks of winter quarter, 2019 (Tuesday, January 8 thru Thursday, February 7)
        1.5 credits

(NoteThis 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. 2015, 2017, 2019 etc).


Meeting Time

T, Th 3:15 to 4:45 FHCRC Day Campus, Weintraub Bldg, room B-072/074

Shuttle from UW:  Departs UW Medical Center at 2:45 for a 3:05 arrival

Shuttle to UW: Departs FHCRC  at 4:58 pm for a 5:10 arrival at UW Medical Center



Barry Stoddard (FHCRC Basic Sciences/UW Biochemistry)  (Weeks 1 to 3)

Phil Bradley (FHCRC Public Health Sciences and Basic Sciences/UW Genome Sciences)  (Week 4)

Abbie Lambert (FHCRC Basic Sciences) (Week 5)


Rationale and Background

While life may have arisen from an RNA-centric origin, and many of the fundamental processes comprising the flow of genetic information 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 four interesting aspects of protein structure and function such as:

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

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

(3) Functional and structural consequences of post-transcriptional and post-translational splicing

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

(5) Protein fold prediction and covariation analyses.

(6) Tandem Repeat Proteins

(7) Combined use of structural information and library design and selection for protein engineering / directed evolution.  Surface Display and flow cytometric analyses and selections.

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.


Assignments and Grading

Students will be graded on any and all of the following:

  1.  In-class assignments or quizzes on the assigned readings, at the discretion of the instructor.
  2.  Demonstration of familiarity with assigned reading during in-class discussion, including oral presentation of answers to discussion questions
  3.  Completion of mid-class protein modeling assignments.


Sessions and Reading Assignments 

All papers will be made available via Dropbox. Readings in (parentheses) are suggested and encouraged but not required; those not in parentheses are assigned for specific discussion and Q/A during class.  Students will be called on to answer questions and/or lead discussion on these.  We limit assigned papers to a couple per session, so please read them thoroughly and be prepared to participate in discussion.   Please don't be 'that person' who comes to a class with no clue about what the papers were about! Dr. Stoddard has been known to make such a person's life uncomfortable in such cases....

Week 1 (January 8 and 10):                                                                 Barry Stoddard


Basics and beyond: visualizing and analyzing protein folds and structures

Shape-shifting and moonlighting proteins


The RCSB Protein Database and the PyMol Visualization Program

Protein Fold Classification and Analysis Servers (PFAM, CATH, SCOP2)

Readings (those in parentheses are suggested only):

1. Burley et al. (2018) "RCSB Protein Data Bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, biotechnology and energy" Nuclei Acids Research doi: 10.1093/nar/gky1004 [Epub ahead of print].  PubMed 30357411.

2.  Tuinstra et al. (2008) “Interconversion between two unrelated protein folds in the lymphotactin native state” PNAS USA 105: 5057 - 5062. PubMed 18364395

(3.  Walden et al. (2006) “Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA” Science 314: 1903 - 1908. PubMed 17185597 )

(4. Kosloff and Kolodny (2008) "Sequence-similar, structure-dissimilar protein pairs in the PDB" Proteins 71: 891 – 902. PubMed 18004789  )

(5. Jeffery (2018) “Protein moonlighting: what is it, and why is it important?” Phil. Trans. Royal Soc. B. PubMed 29203708     PubMedCentral PMC5717523   )


Week 2 (January 15 and 17):                                                               Barry Stoddard


Protein Folds and Design

Protein Modeling

Protein Stabilization and Optimization


Structure Similarity and Superposition Servers (DALI, FATCAT, SUPERPOSE)

Protein Modeling 101 (PSI Protein Model Portal)

Domain identification and homology modeling servers (PHYRE2, SWISSMODEL, I-TASSER)

The PROSS (Protein One Stop Shopping) Stabilization Server (PROSS)

Readings (those in parentheses are suggested only):

1. Kuhlman, Dantas, Ireton, Varani, Stoddard and Baker (2003) ”Design of a novel globular protein fold with atomic-level accuracy” Science 302: 1364 – 1368. PubMed 14631033.

2. Dou et al. (2018) ”De novo design of a fluorescence-activating b-barrel” Nature 561: 485 – 491. PubMed 30209393.

3. Goldenzweig, A. et al. (2016) “Automated structure- and seuqence-based design of proteins for high bacterial expression and stability” Mol. Cell 63 (2): 337 – 346.  PubMed 27425410.

(4.Chandonia et al. (2018) "SCOPe: classification of large macromolecular structures in teh structural classification of proteins -- extended database" Nucleic Acids Research (epub). PubMed 30500919)

(5. Sillitoe et al. (2019) "CATH: expanding the horizons of structure-based functional annotations for genome sequences" Nucleic Acids Research (epub). PubMed 30398663)

(6. El-Gebali et al. (2018) "The Pfam protein families database in 2019" Nucleic Acids Research (epub). PubMed 30357350)

(7. Kelley et al. (2016) "The Phyre2 web portal for protein modeling, prediction and analysis" Nature Protocols 10 (6): 845 - 858) PubMed 25950237  )


Week 3 (January 22 and 24):                                                               Barry Stoddard


Alternative splicing of proteins.

Protein quaternary assembly, cooperativity and allostery


EXPASY Proteomics and Protein Structure Tools

Readings (those in parentheses are suggested only):

1. Garcia et al. (2004) “A conformational switch in the Piccolo C2A domain regulated by alternative splicing” Nature Struct. Mol. Biol. 11: 45 - 53. PubMed 14718922

2. Grinberg et al. (2018) “Novel ATP-con-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit” eLIFE DOI: 10.7554/eLife.31529  PubMed 29388911

(3. Ciragan et al. (2016) “Salt-inducible protein splicing in cis and trans by inteins from extremely halophilic archaea as a novel protein-engineering tool” J. Mol. Biol. 428: 4573 - 4588  PubMed  27720988  )


Week 4 (January 29 and 31):                                                               Phil Bradley


Protein structure prediction

Covariation analyses

Tandem Repeat Proteins



Marks, Debora S., Thomas A. Hopf, and Chris Sander (2012) “Protein Structure Prediction from Sequence Variation.” Nature Biotechnology 30 (11): 1072–80.  PubMed 23138306

Ovchinnikov, Sergey, Hahnbeom Park, Neha Varghese, Po-Ssu Huang, Georgios A. Pavlopoulos, David E. Kim, Hetunandan Kamisetty, Nikos C. Kyrpides, and David Baker  (2017) “Protein Structure Determination Using Metagenome Sequence Data.” Science 355 (6322): 294–98.  PubMed 38104891

Manna, S. (2015) "An overview of pentatricopeptide repeat proteins and their applications" Biochimie 113: 93 - 99. PubMed 25882680


Week 5 (February 5 and 7)                                                                   Abbie Lambert


Structure based protein engineering/directed evolution via randomized library generation and screening.

Protein Surface display

Flow cytometric based selections and analyses.


LAGLIDADG homing endonuclease database and engineering server  (LAHEDES)

Readings (those in parentheses are suggested only):

1.  Werther et al. (2017) “Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity” Nucleic Acids Res. 45: 8621-8634  PubMed 28637173

2. Cherf and Cochran (2015) “Applications of Yeast Surface Display for Protein Engineering” Methods Mol Biol 1319: 155-175.  PubMed 26060074

(3.  Niyonzima et al. (2017) “Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.” Protein Eng Des Sel 30: 503-522.    PubMed 28873986 )

(4. Lambert et al.  (2016) “Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity” Structure 24: 862-73.  PubMed 27133026)

(5. Takeuchi et al. (2011) “Tapping natural reservoirs of homing endonucleases for targeted gene modification.” Proc Natl Acad Sci USA 108: 13077-82.  PubMed 21784983)

(6. Taylor et al. (2012) “LAHEDES: the LAGLIDADG homing endonuclease database and engineering server” Nucleic Acids Res 40 (web server issue): W110-6.  PubMed 22570419)