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Questions and Answers
What does the yeast two-hybrid (Y2H) system primarily utilize to identify protein interactions?
Which of the following is NOT mentioned as a tool for studying protein-protein interactions?
Why are protein-protein interactions considered difficult to predict?
Which biological aspect is significantly influenced by protein-protein interactions?
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What is a characteristic of protein complexes formed through protein-protein interactions?
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In which era was immunoprecipitation primarily used for protein-protein interaction studies?
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Which of the following represents a common domain interaction type in protein-protein interactions?
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What is one of the key reasons for studying protein-protein interactions in the context of biochemistry?
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What is the principle behind gel filtration chromatography?
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What is the primary function of Far western blotting?
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In affinity chromatography, how are His-tagged proteins eluted from the column?
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What type of beads are used in co-immunoprecipitation to capture the protein complex?
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What does protein microarray technology primarily allow researchers to do?
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What is the primary function of the Activation Domain (AD) in the Y2H method?
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Which statement correctly describes the purpose of the yeast two-hybrid assay?
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In the context of protein interactions studied by the Y2H method, what does 'bait' refer to?
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What is one of the disadvantages of the yeast two-hybrid assay mentioned?
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Which of the following interactions is not typically involved in the assembly of virion particles?
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What type of forces primarily mediate the interactions necessary for protein assembly processes?
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Which protein reporter gene is used in the yeast two-hybrid assay to indicate successful transcription?
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What is required for strong couplings in protein-protein interactions?
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Study Notes
Protein-Protein Interactions Lecture Notes
- Course Description: This course provides an introduction to proteomics, genomics, and associated technologies, focusing on protein-protein interactions.
- Date: October 14, 2024
- Instructor: Dr. Manal Abouelwafa, Badr University in Assiut, Biotechnology Department
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Methods for identifying protein-protein interactions:
- Co-immunoprecipitation
- 2-hybrid assay
- Yeast two-hybrid assay
- Far western blot
- Protein microarray
- Fluorescence resonance energy transfer (FRET)
- Confocal microscopy
- Protein microarray
Central Dogma
- DNA → RNA → Protein: The central dogma describes the flow of genetic information from DNA to RNA to protein.
- Transcription: DNA is transcribed into RNA
- Translation: RNA is translated into protein.
- Replication: DNA is replicated
- Reverse transcription: An exception to the basic flow of the central dogma—RNA is used as a template to create DNA.
Gene Expression
- Transcription in Nucleus: DNA in the nucleus is transcribed into messenger RNA (mRNA)
- Splicing: Non-coding regions (introns) of mRNA are removed, leaving coding regions (exons)
- mRNA Export: Mature mRNA is exported to the cytoplasm
- Translation in Cytoplasm: mRNA is translated into a polypeptide chain of amino acids, forming a protein.
- Protein Folding: Polypeptide chains fold into their functional 3D structures.
Splicing
- Intron Removal: The pre-mRNA molecule contains both coding regions (exons) and non-coding regions (introns). Introns are removed during splicing.
- Exon Joining: Exons are joined together to form a mature mRNA molecule containing only coding information.
- GU and AG: These are conserved regions at the intron boundaries.
Introduction to Protein-Protein Interactions
- Importance: Protein-protein interactions are fundamental in many cellular processes. These include cell signaling, localization and trafficking, post-translational modifications, and viral replication.
- Complexity: Predicting protein interactions is challenging. Common patterns are domain-domain interactions and domain interactions with peptides.
- Multicomponent Complexes: Proteins often work together in complexes for specific functions ranging from simple dimers to complex systems like ribosomes.
- Higher Organisms: They tend to have more functional domains, and hence, more complex associations suggesting numerous potential interactions.
Biological Effects of Protein-Protein Interactions
- Inactivation/Suppression: PPIs can inactivate or suppress another protein.
- Novel Binding Sites: PPIs can create new binding sites for small molecules.
- Substrate Specificity: PPIs can modify the specificity of an enzyme for its substrates by altering binding partners
Identifying Protein-Protein Interactions
- Immunoprecipitation: A method used in the pregenomic era to identify protein interactions by using antibodies to isolate proteins.
- Yeast Two-Hybrid (Y2H) Assay: A post-genomic era method involving the cutting of a transcription factor into two parts (DNA-binding domain and activation domain) to find proteins that interact with each other.
- Bait and Prey Proteins: The protein of interest when used to assay other proteins.
Mechanisms of Interaction
- Non-Covalent: These are reversible interactions including van der Waals forces, hydrophobic interactions, electrostatic bonds, and hydrogen bonds.
- Strong Couplings: Accurate force fields are required for strong couplings.
Methods to Study Protein-Protein Interactions
- Gel Filtration: Separates molecules based on size in a porous matrix.
- Far Western Blot: Detects protein-protein interactions using antibodies.
- Affinity Chromatography: Isolates proteins based on their binding to specific ligands.
- Co-Immunoprecipitation: Isolates proteins/protein complexes from a cell extract based on antibody binding.
- Co-crystallization: Used to study protein-protein complexes through crystallography.
- Capillary Electrophoresis: Separates molecules by their charge and size.
- Biosensor: Measures binding interactions through changes in electrical properties.
- FRET (Fluorescence Resonance Energy Transfer): Measures protein-protein interactions by detecting energy transfer between molecules.
- Confocal Microscopy: Visualizes protein interactions in cells.
- 2-hybrid Assay: Protein interaction assay.
- Protein Microarray: High-throughput technique where proteins are arranged on a surface for interaction studies.
- Mass Spectrometry (Maspec): Analysis of protein molecules
- NMR (Nuclear Magnetic Resonance): Technique to study molecular structure and interactions based on magnetic properties of atoms.
- STRING (Search Tool for the Retrieval of Interacting Genes/Proteins): A database and analytical tool used in protein interaction research.
Gel Filtration Chromatography
- Size Exclusion: Molecules are separated based on their size in porous materials.
- Porous Polymers: The chromatography matrix is made from porous material to trap small molecules.
- Proteins Separated: Larger proteins elute first; smaller ones are trapped longer in the porous matrix.
Far Western Blot
- Protein Fractionation: Proteins are separated on SDS-PAGE.
- Blot to Membrane: Proteins are transferred to a membrane such as nitrocellulose or PVDF
- Overlay Protein: The immobilized proteins on the membrane are overlaid by a different protein solution.
- Antibody Detection: Antibodies specific to potentially interacting proteins are used to detect the interaction.
Co-Immunoprecipitation
- Sepharose Beads: Beads coated with protein A are used to isolate protein complexes
- Antibody Binding: Antibodies are designed to bind target proteins.
- Protein Precipitation: The antibody-protein complexes are precipitated with the beads.
- Western Blot (for analysis): The isolated protein complexes are separated and analyzed to confirm the interactions.
Affinity Chromatography
- Nickel/Cobalt Columns: His-tagged proteins can interact with nickel/cobalt immobilized resins.
- Imidazole Elution: The targeted proteins and their related proteins are released from the column by the addition of imidazole, causing a shift in equilibrium.
Protein Microarray
- High-Throughput: Used to track and determine protein interactions.
- Solid Support: Proteins are organized on microscope slides, membranes, beads, or microtitre plates.
- Protein Interactions: Interactions are detected by analyzing binding to each protein spot in the array.
Viral Assembly
- Structural Protein Concentration: Virion assembly begins when enough structural proteins are present in the cell.
- Components Come Together: Nucleocapsid formation is the first stage.
- Whole Particle: The process proceeds until the full virus particle is formed.
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Description
Explore the intricate world of protein-protein interactions in this lecture quiz. Covering methods such as co-immunoprecipitation and the yeast two-hybrid assay, this quiz will test your understanding of key proteomics concepts and the central dogma of molecular biology. Ideal for biotechnology students!
