2025 CHEM 5620 - Protein Chemistry: Final Exam Study Guide | Exams Chemistry

2025 CHEM 5620 - Protein Chemistry: Final Exam Study Guide

Here are 9 key topics to focus on for your upcoming midterm exam. The exam will consist of 25

multiple-choice questions (4 points each) and 4 open-answer questions (25 points each), and you

should be able to complete them within 2 hours. One double-sided letter-sized sheet of notes

may be brought to the exam.

1. Protein Biosynthesis and Unnatural Amino Acid Incorporation

•Components required for translation: mRNA, tRNA, rRNA, ribosomes, aminoacyl-tRNA

synthetases

•Stages of translation: initiation, elongation, termination

•Genetic code expansion and unnatural amino acid (UAA) incorporation

•Orthogonal tRNA/aminoacyl-tRNA synthetase systems

•Site-specific incorporation of unnatural amino acids into proteins and applications

2. Ubiquitination and Protein Degradation in the Context of Auxin Signaling

•Mechanism of ubiquitination (E1, E2, E3 enzymes)

•26S proteasome structure and function

•Auxin Receptor Mechanism in Plants: 1) Auxin, a plant hormone, regulates gene

expression by promoting the degradation of transcriptional repressors known as Aux/IAA

proteins. 2) The F-box protein TIR1 functions as an auxin receptor and is part of the

SCFTIR1 E3 ubiquitin ligase complex. 3) In the presence of auxin, TIR1 binds to Aux/IAA

proteins, leading to their ubiquitination and subsequent degradation by the 26S

proteasome. This degradation releases the repression on auxin-responsive genes,

facilitating various aspects of plant growth and development.

3. Enzyme Catalysis Fundamentals and Directed Evolution of Enzymes

•Definition and general properties of enzymes

•Activation energy and transition state theory

•Gibbs free energy (ΔG) and reaction spontaneity

•Directed Evolution of Enzymes: 1) Principles and methods (e.g., error-prone PCR,

DNA shuffling, continuous directed evolution) 2) Applications in developing enzymes

with novel or enhanced properties (activity, specificity, stability)

•Case study: Evolution of TurboID from ancestral biotin ligase BirA via

directed evolution, and its applications.

4. Molecular Chaperones and Protein Folding Diseases

•Role of molecular chaperones in protein folding and proteostasis.

•Molecular chaperones as facilitators of protein evolution (e.g., chaperonin-assisted

directed evolution of enzymes).

•Protein misfolding-associated degenerative diseases and disease

mechanisms: Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases.

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2025 CHEM 5620 - Protein Chemistry: Final Exam Study Guide Here are 9 key topics to focus on for your upcoming midterm exam. The exam will consist of 25 multiple-choice questions (4 points each) and 4 open-answer questions (25 points each), and you should be able to complete them within 2 hours. One double-sided letter-sized sheet of notes may be brought to the exam.

1. Protein Biosynthesis and Unnatural Amino Acid Incorporation - Components required for translation: mRNA, tRNA, rRNA, ribosomes, aminoacyl-tRNA synthetases - Stages of translation: initiation, elongation, termination - Genetic code expansion and unnatural amino acid (UAA) incorporation - Orthogonal tRNA/aminoacyl-tRNA synthetase systems - Site-specific incorporation of unnatural amino acids into proteins and applications 2. Ubiquitination and Protein Degradation in the Context of Auxin Signaling - Mechanism of ubiquitination (E1, E2, E3 enzymes) - 26S proteasome structure and function - Auxin Receptor Mechanism in Plants: 1) Auxin, a plant hormone, regulates gene expression by promoting the degradation of transcriptional repressors known as Aux/IAA proteins. 2) The F-box protein TIR1 functions as an auxin receptor and is part of the SCFTIR1^ E3 ubiquitin ligase complex. 3) In the presence of auxin, TIR1 binds to Aux/IAA proteins, leading to their ubiquitination and subsequent degradation by the 26S proteasome. This degradation releases the repression on auxin-responsive genes, facilitating various aspects of plant growth and development. 3. Enzyme Catalysis Fundamentals and Directed Evolution of Enzymes - Definition and general properties of enzymes - Activation energy and transition state theory - Gibbs free energy (ΔG) and reaction spontaneity - Directed Evolution of Enzymes: 1) Principles and methods (e.g., error-prone PCR, DNA shuffling, continuous directed evolution) 2) Applications in developing enzymes with novel or enhanced properties (activity, specificity, stability) - Case study: Evolution of TurboID from ancestral biotin ligase BirA via directed evolution, and its applications. 4. Molecular Chaperones and Protein Folding Diseases - Role of molecular chaperones in protein folding and proteostasis. - Molecular chaperones as facilitators of protein evolution (e.g., chaperonin-assisted directed evolution of enzymes). - Protein misfolding-associated degenerative diseases and disease mechanisms: Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases.

5. Protein Phase Separation and its Biological Significance - Fundamental principles of phase transitions and phase separation. - Molecular mechanisms underlying intracellular LLPS: roles of multivalent proteins, intrinsically disordered regions (IDRs) in proteins, and RNA. - Biological functions and implications of LLPS in cell biology (e.g., stress granules, transcriptional regulation, chromatin organization). - Dysregulation of protein phase separation in diseases, including neurodegenerative diseases and cancer. 6. Protein Evolution: Mechanisms, Constraints, and Applications - Basic concepts of protein evolution: homologs, paralogs, orthologs, and phylogenetic analysis. - Molecular mechanisms driving protein evolution: mutations, natural selection, genetic drift, gene duplication, horizontal gene transfer. - Evolutionary constraints shaping protein structure, function, stability, and coevolution. - Case study: Evolutionary pathways of antifreeze proteins in fish. 7. Bioorthogonal Chemistry and Fluorescent Labeling and Imaging - Definition, principles, and key reaction types (CuAAC, SPAAC, iEDDA). - Site-specific protein labeling strategies for studying biological processes, such as enzymatic labeling (e.g., sortase-mediated ligation). - Applications and mechanisms of super-resolution microscopy (e.g., photoactivated localization microscopy (PALM)). 8. Protein-Small Molecule and Protein-Protein Interactions: Mechanisms and Methods - Fundamentals of protein-small molecule and protein-protein interactions: non- covalent forces (hydrogen bonds, hydrophobic interactions, Van der Waals forces, ionic interactions). - Thermodynamics and kinetics of molecular interactions: Gibbs free energy (ΔG), binding affinity, entropy vs. enthalpy-driven interactions. - Experimental techniques to characterize interactions: Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC). - In vivo techniques for studying protein interactions: Yeast-Two Hybridization, Bimolecular Fluorescence Complementation (BiFC), and TurboID. 9. Protein Engineering in Biotechnology Applications - Antibody-Drug Conjugates (ADCs): These involve combining monoclonal antibodies with cytotoxic payloads to create targeted cancer therapies. Key engineering considerations include site-specific conjugation, stability, linker chemistry, and payload potency.

2025 CHEM 5620 - Protein Chemistry: Final Exam Study Guide, Exams of Chemistry

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