Biotech Chapter 5

Introduction to Studying Proteins

• Proteins are crucial to biotechnology.
• Protein's three-dimensional structure is essential for function.
• Researchers study protein structure and amino acid sequences to create protein products.

Learning Outcomes

• Describe protein structure, emphasizing the role of amino acid R-groups.
• Explain protein synthesis (transcription and translation).
• Discuss the role of naturally occurring and recombinant proteins in biotechnology.
• Classify proteins based on their function (structural, antibody, enzyme).
• Differentiate between antibodies and antigens.
• Distinguish among enzyme classes and reaction conditions impacting enzyme activity.
• Summarize polyacrylamide gel electrophoresis (PAGE) and its use in studying proteins.

5.1 The Structure and Function of Proteins

• Virtually all biotechnology products involve proteins.
• Proteins' three-dimensional structure defines their function.
• To develop a protein product, researchers study protein structure and amino acid sequence.
• X-ray crystallography is a technique that reveals protein structure.

Protein Molecule Structure

• Proteins consist of one or more folded peptide chains made of amino acids.
• Human salivary amylase is an example, with over 500 amino acids.

Amino Acid Structure

• Twenty types of amino acids are found in proteins.
• Most proteins include hundreds of amino acids.
• Amino acids share a common core structure, differing only in their R-group.

Protein 3-D Structure

• Primary structure: the sequence of amino acids in a protein.
• Protein folding is influenced by interactions between amino acids.
• Secondary structure includes alpha helices and beta-pleated sheets.
• Tertiary structure defines the overall 3D protein shape.
• Quaternary structure results from multiple polypeptide interactions.

Impact of Structure on Protein Function

• The precise shape of a protein determines its interaction with other molecules.
• Antibodies recognize and bind foreign molecules for removal.
• Antibodies have a variable region for binding and a constant region for recognition.

5.1 Vocabulary

• X-ray crystallography: technique to determine 3D protein structure.
• Polar: contains both positive and negative charges.
• Primary structure: order and type of amino acids.
• Secondary structure: structure resulting from hydrogen bonds (e.g., alpha helix, beta sheet).
• Tertiary structure: structure formed from interactions involving "R" groups and hydrogen bonds.
• Quaternary structure: structure formed by multiple polypeptide interactions.
• Glycoprotein: protein with added sugar groups.
• Glycosylated: descriptive of a molecule with added sugar groups.
• CD4 cells: human white blood cells with CD4 protein on their surface.
• Antigens: foreign proteins/molecules targeted by antibodies
• Epitope: specific region on a molecule recognized by an antibody.
• ELISA: technique to measure protein/antibody amounts.
• Monoclonal antibody: antibody directed against a single epitope.
• Hybridoma: hybrid cell generating monoclonal antibodies.

5.2 The Production of Proteins

• Proteins were once exclusively produced in cells.
• Small polypeptide chains are now produced in labs using protein synthesizers.
• Protein synthesis is a two-step process: transcription and translation.
• Transcription: Converting DNA code to mRNA.
• Translation: Converting mRNA code into amino acid sequence.
• Protein synthesis occurs constantly within cells.

Transcription and Translation

• DNA's genetic code needs to be converted to mRNA.
• Protein synthesis (transcription and translation) involves crucial steps and molecules.
• The process converts the genetic code (DNA) into a protein.
• mRNA copies the genetic information from DNA.

### Protein Synthesis in Prokaryotes

•  Protein synthesis in prokaryotes follows similar principles to that in eukaryotes.
• This process involves a circular single strand of DNA and various molecules like mRNA and tRNA.

5.2 Vocabulary

• Protein synthesis: generation of new proteins from amino acid building blocks.
• Transcription: Process of deciphering DNA code to RNA code.
• Codon: Set of three nucleotides in mRNA coding for a specific amino acid.
• Translation: Process of decoding mRNA into amino acid sequence.
• tRNA: Type of RNA that brings amino acids to the ribosome.
• Peptidyl transferase: Enzyme in ribosomes connecting amino acids.
• Phosphorylation: Addition of phosphate groups to molecules.
• Cleavage: Process of splitting a polypeptide into smaller strands.
• Taq polymerase: DNA synthesis enzyme resistant to high temperatures.

5.3 Enzymes: Protein Catalysts

• Enzymes are proteins acting as catalysts for cellular reactions.
• Enzymes are involved in practically every biochemical reaction.
• Enzymes act on substrates fitting specific active site shapes. This interaction can be described by theories such as lock and key and induced fit model.
• Enzymes' catalytic behaviour depends on different factors.

Enzyme Groups and Functions

• Hydrolases: split substrates using water.
• Lyases: split substrates without water.
• Transferases: move chemical groups between molecules.
• Isomerases: rearrange substrate molecules.
• Oxidoreductases: transfer electrons.
• Synthetases: combine molecules.
• Examples for each enzyme group are provided to illustrate their functions.

Factors That Affect Enzyme Activity

• Substrate concentration: more substrate, faster reaction.
• Temperature: enzymes have optimal temperatures.
• pH: enzymes have optimal pH values.

Optimum pH

• Each enzyme has an optimal pH range for maximum activity.
• pH outside of the optimal range usually reduces enzyme activity.

5.3 Vocabulary

• Substrate: molecule acted upon by an enzyme.
• Cofactors: Atoms/molecules required for enzyme activity.
• Lock and Key Model: Enzyme and substrate fit perfectly.
• Induced Fit Model: Enzyme changes shape to fit substrate.
• Optimum temperature: Temperature for optimal enzyme activity.
• Denaturation: Loss of protein shape (3D structure).
• Optimum pH: pH for maximum enzyme activity.

5.4 Studying Proteins - PAGE

• Protein samples are separated on a polyacrylamide gel using a technique called polyacrylamide gel electrophoresis (PAGE).
• Vertical PAGE gel boxes are similar in operation to horizontal PAGE gel boxes.
• Protein size is determined using protein standards/markers.
• Smaller proteins move faster through the gel.

PAGE – Vertical Gel Electrophoresis

• Vertical PAGE gel boxes have a similar design to horizontal PAGE gel boxes.
• Gel cassettes are snapped or screwed into the box.
• Running buffer covers the wells and the front of the gel cassette.
• Electric current moves negatively charged protein samples down the gel towards the positive electrode.

PAGE with Standards

• The smaller the protein, the faster it migrates through the gel.
• Protein sizing markers/standards allow for the size determination of unknown samples by comparison.
• Protein molecular weight is often represented in kilodaltons (kDa).

Staining Methods

• Silver stain is a much more sensitive staining method to visualize proteins and DNA when concentrations are low.

5.4 Vocabulary

• PAGE: Polyacrylamide gel electrophoresis - Separation of proteins and DNA.
• Coomassie Blue: Dye that stains proteins, making them visible on gel.
• Silver stain: More sensitive stain for protein visualization than Coomassie Blue.

5.5 Applications of Protein Analysis

• Studying protein profiles of cells/tissues provides insights into cellular function.
• Protein structure relates to protein function.
• Understanding proteins provides insights into cellular mechanisms, evolution, and taxonomic relationships.
• The National Center for Biotechnology Information (NCBI) hosts databases and tools for protein and DNA analysis.

5.5 Vocabulary

• Taxonomic relationships: how species are related through evolution.
• Biomanufacturing: industry focusing on protein production through biotechnology.

This quiz explores the critical role of proteins in biotechnology and their three-dimensional structures essential for function. Participants will learn about protein synthesis, classification, and techniques like polyacrylamide gel electrophoresis. Enhance your understanding of how proteins impact various biotechnological applications.