Introduction to Introduction to Studying ProteinsStudying Proteins

Chapter 5Chapter 5

ObjectivesObjectives

Describe the main roles that proteins play Describe the main roles that proteins play in organisms.in organisms.

Describe the process by which proteins are Describe the process by which proteins are assembled.assembled.

Describe how amino acids differ from one Describe how amino acids differ from one another.another.

Describe the process by which an enzyme Describe the process by which an enzyme functions and list factors that influence functions and list factors that influence their ability to work properly.their ability to work properly.

Describe the process of SDS-PAGE.Describe the process of SDS-PAGE.

Why study proteins?Why study proteins?

Proteins in organisms canProteins in organisms can– Serve as structural components in cellsServe as structural components in cells

i.e. collageni.e. collagen

– Act as enzymesAct as enzymes Carry out essential chemical reactionsCarry out essential chemical reactions i.e. catalasei.e. catalase

Enzymes are of extreme interest due Enzymes are of extreme interest due to application in medicine and to application in medicine and potential commercial use.potential commercial use.

Protein StructureProtein Structure Protein structure is Protein structure is

critical in determining critical in determining what the protein can do.what the protein can do.

Proteins are a type of Proteins are a type of macromolecule that are macromolecule that are assembled from assembled from subunits called amino subunits called amino acids.acids.– Usually tens or hundreds Usually tens or hundreds

of amino acids chained of amino acids chained together.together.

All organisms build their All organisms build their proteins from a pool of proteins from a pool of 20 amino acids.20 amino acids.

Protein StructureProtein Structure

All amino acids All amino acids have the same have the same basic structure.basic structure.

What separates What separates one amino acid one amino acid from another?from another?– ““R group”R group”

Charged (+/-)Charged (+/-) PolarPolar UnchargedUncharged

tyrosine (tyr) lysine (lys) glutamate (glu) glycine (gly)

valine (val) phenylalanine (phe) methionine (met) proline (pro)

Protein StructureProtein Structure

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

reactionreaction

Protein StructureProtein Structure

Ways protein Ways protein structure can be structure can be represented:represented:– Primary structurePrimary structure

Linear sequence of Linear sequence of amino acids.amino acids.

gly-ala-val-progly-ala-val-pro

– Secondary structureSecondary structure Three dimensional Three dimensional

structure of either structure of either alpha helix or beta alpha helix or beta sheet.sheet.

Protein StructureProtein Structure

Ways protein structure Ways protein structure can be represented:can be represented:– Tertiary structureTertiary structure

Final three dimensional Final three dimensional shape a polypeptide shape a polypeptide takes.takes.

– Quaternary structureQuaternary structure Proteins with more than Proteins with more than

one polypeptide chain.one polypeptide chain.– i.e. collagen or i.e. collagen or

hemoglobinhemoglobin

Protein SynthesisProtein Synthesis

Protein production is controlled by a Protein production is controlled by a variety of organelles in a cell.variety of organelles in a cell.

Cells are very precise in determining Cells are very precise in determining when and how much of a protein is when and how much of a protein is needed.needed.

In medicine and industry In medicine and industry biotechnologist have found ways to biotechnologist have found ways to manipulate cells into making large manipulate cells into making large numbers of specific proteins.numbers of specific proteins.

Protein SynthesisProtein Synthesis

The instructions for how to make a protein The instructions for how to make a protein reside in the cells DNA, found in the reside in the cells DNA, found in the nucleus.nucleus.

The machinery for making the protein, The machinery for making the protein, ribosomes, are found outside the nucleus.ribosomes, are found outside the nucleus.

Protein SynthesisProtein Synthesis

Getting the Getting the instructions from instructions from the DNA to the the DNA to the ribosomes involves ribosomes involves transcription.transcription.– Reminder that bases Reminder that bases

used for producing used for producing RNA are A, U, C, G.RNA are A, U, C, G.

The mRNA The mRNA produced leaves produced leaves the nucleus.the nucleus.

Protein SynthesisProtein Synthesis

The actual protein The actual protein product is product is produced during produced during translation.translation.

Ribosomes use Ribosomes use mRNA and tRNA mRNA and tRNA molecules to molecules to assemble the a.a’s assemble the a.a’s in the correct in the correct sequence.sequence.

Protein SynthesisProtein Synthesis

The order of the The order of the amino acids is amino acids is determined by the determined by the three base sequences three base sequences of the mRNA.of the mRNA.– CodonsCodons

tRNA molecules that tRNA molecules that carry the amino acids carry the amino acids have complementary have complementary anti-codonsanti-codons

Protein SynthesisProtein Synthesis

Protein SynthesisProtein Synthesis

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

EnzymesEnzymes

Enzymes represent one important type Enzymes represent one important type or group of proteins.or group of proteins.

Cells carry out millions of chemical Cells carry out millions of chemical reactions every second.reactions every second.– Without enzymes they would not occur Without enzymes they would not occur

fast enough to meet the needs of the cells.fast enough to meet the needs of the cells. The amount and type of enzyme The amount and type of enzyme

produced are tightly controlled by the produced are tightly controlled by the cell.cell.

EnzymesEnzymes How do enzymes work?How do enzymes work?

– When an enzyme speeds up a reaction, the participating When an enzyme speeds up a reaction, the participating reactants are called the enzyme’s substrates.reactants are called the enzyme’s substrates.

– Enzymes help speed up reactions that otherwise would occur Enzymes help speed up reactions that otherwise would occur on there own.on there own.

– The end result of the reactions (degradation or synthesis) is The end result of the reactions (degradation or synthesis) is the formation of product(s).the formation of product(s).

– Enzymes themselves are NOT used up in the reaction.Enzymes themselves are NOT used up in the reaction.– Some enzymes require cofactors.Some enzymes require cofactors.

EnzymesEnzymes

Factors that Factors that influence how influence how enzymes work.enzymes work.– TemperatureTemperature– pHpH

Swings outside Swings outside specified ranges specified ranges can result in can result in denaturation of the denaturation of the enzyme.enzyme.

EnzymesEnzymes Enzymes are divided in six categories based on Enzymes are divided in six categories based on

their function.their function.– Hydrolases…..break chemical bondHydrolases…..break chemical bond– Lyases…… break chemical bondLyases…… break chemical bond– Transferases….transfer of a functional group from one Transferases….transfer of a functional group from one

molec to anothermolec to another– Isomerases….. catalyses interconversion of isomers Isomerases….. catalyses interconversion of isomers – Oxidoreductases…. catalyzes transfer of electrons from Oxidoreductases…. catalyzes transfer of electrons from

one molecule to anotherone molecule to another– Synthetases….making new molecSynthetases….making new molec

Note that enzymes are normally denoted by Note that enzymes are normally denoted by having the suffix “-ase” added to their name.having the suffix “-ase” added to their name.– I.e. sucraseI.e. sucrase

Studying ProteinsStudying Proteins

Companies that produce protein Companies that produce protein products or study proteins must be products or study proteins must be able to:able to:– Separate the protein of interest.Separate the protein of interest.– Determine that amount of protein present.Determine that amount of protein present.

Characteristics of proteins that make it Characteristics of proteins that make it possible to achieve either one of both possible to achieve either one of both points above:points above:– Overall charge, size, shape, and solubilityOverall charge, size, shape, and solubility

Studying ProteinsStudying Proteins

Spotlight: SDS-PAGESpotlight: SDS-PAGE– Gel electrophoresis allows for the Gel electrophoresis allows for the

separation of proteins based on charge, separation of proteins based on charge, size, and shape.size, and shape.

– Polyacrylamide gel electrophoresis is Polyacrylamide gel electrophoresis is utilized (PAGE).utilized (PAGE). Allows for better resolutionAllows for better resolution 4-18% gels most commonly used4-18% gels most commonly used

– Higher concentration for smaller proteinsHigher concentration for smaller proteins When protein size unknown gradient gels can When protein size unknown gradient gels can

be used.be used.– Less concentrated at the top than the bottom.Less concentrated at the top than the bottom.

Studying ProteinsStudying Proteins

Spotlight: SDS-PAGESpotlight: SDS-PAGE– Use of sodium Use of sodium

dodecyl sulfate (SDS)dodecyl sulfate (SDS) Denatures proteins into Denatures proteins into

polypeptide strandspolypeptide strands Gives each polypeptide Gives each polypeptide

strand an overall strand an overall negative chargenegative charge

Proteins studied are Proteins studied are strictly being strictly being separated by size.separated by size.

Studying ProteinsStudying Proteins

Spotlight: SDS-PAGESpotlight: SDS-PAGE– Visualization of the Visualization of the

proteins in the gel.proteins in the gel. Coomassie BlueCoomassie Blue

– Milligram amounts of Milligram amounts of protein.protein.

Silver stainSilver stain– Microgram amounts of Microgram amounts of

protein.protein.

– Size of unknown bands Size of unknown bands can be determined can be determined from comparison to the from comparison to the protein molecular protein molecular weight standard.weight standard.

Homework 5Homework 5

Review 5.1Review 5.1– Questions 1, 2, 3Questions 1, 2, 3

Review 5.2Review 5.2– Questions 1, 2, 3Questions 1, 2, 3

Review 5.3Review 5.3– Questions 1, 2Questions 1, 2

Review 5.4Review 5.4– Questions 1, 2Questions 1, 2

Think like BiotechThink like Biotech– Questions: 2, 3, 5, 6, 7Questions: 2, 3, 5, 6, 7

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