Introduction to Studying Proteins PDF

Introduction to Studying Proteins Chapter 5  Learning Outcomes ! Describe the structure of proteins, including the significance of amino acid R-groups and their impact on the three-dimensional structure of proteins. ! Explain the steps of transcription and translation in protein sy...

Introduction to Studying Proteins Chapter 5  Learning Outcomes ! Describe the structure of proteins, including the significance of amino acid R-groups and their impact on the three-dimensional structure of proteins. ! Explain the steps of transcription and translation in protein synthesis. ! Discuss the role of naturally occurring proteins and recombinant proteins in biotechnology. ! Differentiate proteins that function as part of structure, as antibodies, and as enzymes. ! Describe the structure of antibodies and explain the relationship between antibodies and antigens. ! Discriminate among the classes of enzymes and discuss the effect of reaction conditions on enzyme activity. ! Summarize polyacrylamide gel electrophoresis and identify its usefulness for studying proteins.  5.1 The Structure and Function of Proteins Virtually all biotechnology products have something to do with proteins. The three-dimensional structure of a protein is critical for its function. To produce a protein product, researchers must learn about the structure and function of the protein, as well as the amino acid Computer-generated model of the structure of sequence acetylcholinesterase, an enzyme that breaks down molecules to regulate nerve impulses junction between nerve cells. Data collected with x-ray crystallography.  Protein Molecule Structure Proteins are one or more folded, functional peptide chains composed of amino acids Human Salivary Amylase Computer-generated space-filling model of human salivary amylase. There are over 500 amino acids in the single peptide chain of enzyme.  Amino Acid Structure Twenty different kinds of amino acids are found in proteins Most proteins contain tens of hundreds of amino acids  Protein 3-D Structure Peptide Chain Primary structure = which amino acids in which order in the peptide chain Protein folding due to interactions between amino acids in and between peptide chains Alpha helixes and beta pleated sheets fold the peptide chain Alpha Helix Beta-pleated Sheets Impact of Structure on Protein Function For an HIV particle to recognize, attach, and infect a T-helper cell, the gp210 structure must be a precise shape and must exactly match its human cell membrane receptors Function and Structure of Antibodies Recognize and bind “foreign” proteins or other molecules (antigens) for removal from the body  Section 5.1 Vocabulary X-ray crystallography – a technique used to determine the three-dimensional structure of a protein Polar – the chemical characteristic of containing both a positive and negative charge on opposite sides of a molecule Primary structure – the order and type of amino acids found in a polypeptide chain Secondary structure – the structure of a protein (alpha helix and beta sheets) that results from hydrogen bonding Tertiary structure – the structure of a protein that results from several interactions, the presence of charged or uncharged “R” groups, and hydrogen bonding Quaternary structure – the structure of a protein resulting from the association of two or more polypeptide chains Glycoprotein - a protein which has had sugar groups added to it Glycosylated – descriptive of molecules to which sugar groups have been added CD4 cells – the human white blood cells, which contain the cell surface recognition protein CD4  Section 5.1 Vocabulary CD4 cells – the human white blood cells, which contain the cell surface recognition protein CD4 Antigens – the foreign proteins or molecules that are the target of binding by antibodies Epitope – the specific region on a molecule that an antibody binds to ELISA – short for enzyme-linked immunosorbent assay, a technique that measures the amount of protein or antibody in a solution Monoclonal antibody – a type of antibody that is directed against a single epitope Hybridoma – a hybrid cell used to generate monoclonal antibodies that results from the fusion of immortal tumor cells with specific antibody-producing white blood cells (B-cells)  5.2 The Production of Proteins Until recently, proteins could only be made in cells. Now, small polypeptide chains can be made in the lab on protein synthesizers. Overview of Protein Synthesis Protein Synthesis in a Eukaryotic Cell In a eukaryotic cell, DNA (chromosomes) is in the nucleus. The mRNA transcripts carry the DNA code to the ribosomes, which translate the code into a strand of amino acids. Protein synthesis occurs continuously through a cell’s life  Transcription and Translation Protein synthesis is a two-step process: Step 1: Transcription Genetic code (on DNA) must be rewritten onto a messenger molecule (mRNA)  Step 2: Translation mRNA strand with nucleotide code is rendered into a sequence of amino acids that fold into a functional protein Protein Synthesis in Prokaryotes  Section 5.2 Vocabulary Protein synthesis – the generation of new proteins from amino acid subunits; in the cell, it includes transcription and translation Transcription – the process of deciphering a DNA nucleotide code and converting it into RNA nucleotide code; the RNA carries the genetic message to a ribosome for translation into a protein code Codon – a set of three nucleotides on a strand of mRNA that codes for a particular amino acid in a protein chain Translation – the process of reading a mRNA nucleotide code and converting it into a sequence of amino acids tRNA – a type of ribonucleic acid (RNA) that shuttles amino acids into the ribosome for protein synthesis Peptidyl transferase – an enzyme found in the ribosome that builds polypeptide chains by connecting amino acids into long chains through peptide bonds Phosphorylation – adding phosphate groups Cleavage – process of splitting the polypeptide into two or more strands Taq polymerase – a DNA synthesis enzyme that can withstand the high temperatures used in PCR  5.3 Enzymes: Protein Catalysts Enzymes are proteins that act as catalysts Enzymes are involved in virtually every reaction in a cell Many companies have focused on producing enzymes for sale Enzymes and Their Substrates The molecules upon which enzymes act are called substrates Substrate fits into enzyme’s active site by either: > lock and key model > induced fit model Adenosine deaminase (green molecule) binds one of its substrates, a purine nucleoside (red). Zinc ions (purple) are a required cofactor for the enzyme’s activity. Adenosine deaminase is necessary to fight infections. Enzyme Groups and Functions Enzymes are divided into six categories based on their function. Factors That Affect Enzyme Activity Amount of substrate in a solution Temperature of a reaction Acidity or alkalinity Optimum pH  Section 5.3 Vocabulary Substrate – the molecule that an enzyme acts on Cofactors – an atom or molecule that an enzyme requires to function Lock and key model – a model used to describe how enzymes function, in which the enzyme and substrate make an extract molecular fit at the active site, triggering catalysis Induced fit model – a model used to describe how enzymes function, in which a substrate squeezes into an active site and induces the enzyme’s activity Optimum temperature – the temperature at which an enzyme achieves maximum activity Denaturation – the process in which proteins lose their conformation or three-dimensional shape Optimum pH – the pH at which an enzyme achieves maximum activity 5.4 Studying Proteins - PAGE A technician loads protein samples on a vertical gel. Vertical gel boxes operate in a fashion similar to horizontal gel boxes. PAGE = Vertical Gel Electrophoresis. Although vertical gel boxes vary from one manufacturer to another, all are basically of the same design. The gel cassettes are snapped or screwed in place (right). Running buffer is added behind the gel, covering the wells. Buffer is poured in the front of the gel cassette to cover the front opening. When the top is placed on the box (left) and the power is turned on, electricity flows from the top (negative charge) to bottom (positive charge). Negatively charged samples move down the gel toward the positive electrode. PAGE with Standards The smaller the peptide chain, the faster it moves through the gel. Protein sizing standards (markers) can be used to determine the size of unknown samples. Proteins sizes are reported in kilodaltons (kDa). Silver stain is much more sensitive than Coomassie® Blue. When samples have low concentrations of protein or DNA, silver-staining is the method of choice.  Section 5.4 Vocabulary PAGE – short for polyacrylamide gel electrophoresis, a process in which proteins and small DNA molecules are separated by electrophoresis on vertical gels made of the synthetic polymer, polyacrylamide Coomassie® Blue – a dye that stains proteins blue and allows them to be visualized Silver stain – a stain used for visualizing proteins  5.5 Applications of Protein Analysis Protein profile of cells and tissues A protein’s structure can help explain its function Study chemical processes in cells, evolution & taxonomic relationships The National Center for Biotechnology (NCBI) is a collection of databases and analysis tools used for biomedical and biotechnology research. Protein and DNA sequences are stored on databases and shared among scientists at the NCBI site.  Section 5.5 Vocabulary Taxonomic relationships – how species are related to one another in terms of evolution Biomanufacturing – the industry focusing on the production of proteins and other products created by biotechnology