Introduction to Studying Proteins Chapter 5

Objectives Describe the main roles that proteins play in organisms. Describe the main roles that proteins play in organisms. Describe the process by which proteins are assembled. Describe the process by which proteins are assembled. Describe how amino acids differ from one another. Describe how amino acids differ from one another. Describe the process by which an enzyme functions and list factors that influence their ability to work properly. Describe the process by which an enzyme functions and list factors that influence their ability to work properly. Describe the process of SDS-PAGE. Describe the process of SDS-PAGE.

Why study proteins? Proteins in organisms can Proteins in organisms can –Serve as structural components in cells  i.e. collagen –Act as enzymes  Carry out essential chemical reactions  i.e. catalase Enzymes are of extreme interest due to application in medicine and potential commercial use. Enzymes are of extreme interest due to application in medicine and potential commercial use.

Protein Structure Protein structure is critical in determining what the protein can do. Protein structure is critical in determining what the protein can do. Proteins are a type of macromolecule that are assembled from subunits called amino acids. Proteins are a type of macromolecule that are assembled from subunits called amino acids. –Usually tens or hundreds of amino acids chained together. All organisms build their proteins from a pool of 20 amino acids. All organisms build their proteins from a pool of 20 amino acids.

Protein Structure All amino acids have the same basic structure. All amino acids have the same basic structure. What separates one amino acid from another? What separates one amino acid from another? –“R group”  Charged (+/-)  Polar  Uncharged

tyrosine (tyr)lysine (lys)glutamate (glu)glycine (gly) valine (val)phenylalanine (phe)methionine (met)proline (pro)

Protein Structure How is one amino acid connected to another to form a polypeptide chain? How is one amino acid connected to another to form a polypeptide chain? –Condensation reaction

Protein Structure Ways protein structure can be represented: Ways protein structure can be represented: –Primary structure  Linear sequence of amino acids.  gly-ala-val-pro –Secondary structure  Three dimensional structure of either alpha helix or beta sheet.

Protein Structure Ways protein structure can be represented: Ways protein structure can be represented: –Tertiary structure  Final three dimensional shape a polypeptide takes. –Quaternary structure  Proteins with more than one polypeptide chain. –i.e. collagen or hemoglobin

Protein Synthesis Protein production is controlled by a variety of organelles in a cell. Protein production is controlled by a variety of organelles in a cell. Cells are very precise in determining when and how much of a protein is needed. Cells are very precise in determining when and how much of a protein is needed. In medicine and industry biotechnologist have found ways to manipulate cells into making large numbers of specific proteins. In medicine and industry biotechnologist have found ways to manipulate cells into making large numbers of specific proteins.

Protein Synthesis The instructions for how to make a protein reside in the cells DNA, found in the nucleus. The instructions for how to make a protein reside in the cells DNA, found in the nucleus. The machinery for making the protein, ribosomes, are found outside the nucleus. The machinery for making the protein, ribosomes, are found outside the nucleus.

Protein Synthesis Getting the instructions from the DNA to the ribosomes involves transcription. Getting the instructions from the DNA to the ribosomes involves transcription. –Reminder that bases used for producing RNA are A, U, C, G. The mRNA produced leaves the nucleus. The mRNA produced leaves the nucleus.

Protein Synthesis The actual protein product is produced during translation. The actual protein product is produced during translation. Ribosomes use mRNA and tRNA molecules to assemble the a.a’s in the correct sequence. Ribosomes use mRNA and tRNA molecules to assemble the a.a’s in the correct sequence.

Protein Synthesis The order of the amino acids is determined by the three base sequences of the mRNA. The order of the amino acids is determined by the three base sequences of the mRNA. –Codons tRNA molecules that carry the amino acids have complementary anti-codons tRNA molecules that carry the amino acids have complementary anti-codons

Protein Synthesis

Once a polypeptide chain forms it can be further modified (posttranslational modifications). Once a polypeptide chain forms it can be further modified (posttranslational modifications). –Addition of important chemical groups. –Phosphorylation –Cleavage

Enzymes Enzymes represent one important type or group of proteins. Enzymes represent one important type or group of proteins. Cells carry out millions of chemical reactions every second. Cells carry out millions of chemical reactions every second. –Without enzymes they would not occur fast enough to meet the needs of the cells. The amount and type of enzyme produced are tightly controlled by the cell. The amount and type of enzyme produced are tightly controlled by the cell.

Enzymes How do enzymes work? How do enzymes work? –When an enzyme speeds up a reaction, the participating reactants are called the enzyme’s substrates. –Enzymes help speed up reactions that otherwise would occur on there own. –The end result of the reactions (degradation or synthesis) is the formation of product(s). –Enzymes themselves are NOT used up in the reaction. –Some enzymes require cofactors.

Enzymes Factors that influence how enzymes work. Factors that influence how enzymes work. –Temperature –pH Swings outside specified ranges can result in denaturation of the enzyme. Swings outside specified ranges can result in denaturation of the enzyme.

Enzymes Enzymes are divided in six categories based on their function. Enzymes are divided in six categories based on their function. –Hydrolases…..break chemical bond –Lyases…… break chemical bond –Transferases….transfer of a functional group from one molec to another –Isomerases….. catalyses interconversion of isomers –Oxidoreductases…. catalyzes transfer of electrons from one molecule to another –Synthetases….making new molec Note that enzymes are normally denoted by having the suffix “-ase” added to their name. Note that enzymes are normally denoted by having the suffix “-ase” added to their name. –I.e. sucrase

Studying Proteins Companies that produce protein products or study proteins must be able to: Companies that produce protein products or study proteins must be able to: –Separate the protein of interest. –Determine that amount of protein present. Characteristics of proteins that make it possible to achieve either one of both points above: Characteristics of proteins that make it possible to achieve either one of both points above: –Overall charge, size, shape, and solubility

Studying Proteins Spotlight: SDS-PAGE Spotlight: SDS-PAGE –Gel electrophoresis allows for the separation of proteins based on charge, size, and shape. –Polyacrylamide gel electrophoresis is utilized (PAGE).  Allows for better resolution  4-18% gels most commonly used –Higher concentration for smaller proteins  When protein size unknown gradient gels can be used. –Less concentrated at the top than the bottom.

Studying Proteins Spotlight: SDS-PAGE Spotlight: SDS-PAGE –Use of sodium dodecyl sulfate (SDS)  Denatures proteins into polypeptide strands  Gives each polypeptide strand an overall negative charge  Proteins studied are strictly being separated by size.

Studying Proteins Spotlight: SDS-PAGE Spotlight: SDS-PAGE –Visualization of the proteins in the gel.  Coomassie Blue –Milligram amounts of protein.  Silver stain –Microgram amounts of protein. –Size of unknown bands can be determined from comparison to the protein molecular weight standard.

Homework 5 Review 5.1 Review 5.1 –Questions 1, 2, 3 Review 5.2 Review 5.2 –Questions 1, 2, 3 Review 5.3 Review 5.3 –Questions 1, 2 Review 5.4 Review 5.4 –Questions 1, 2 Think like Biotech Think like Biotech –Questions: 2, 3, 5, 6, 7

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