ProteinsProteins

ProteinsProteins

Consists of C, H, O, and NConsists of C, H, O, and N Uses: develop muscle, hair, skin, vital Uses: develop muscle, hair, skin, vital

organs, enzymes, some hormones. Last organs, enzymes, some hormones. Last resort energy.resort energy.

Basic unit of structure is an Amino Acid:Basic unit of structure is an Amino Acid: R is different for eachR is different for each

• There are 20 different types of Amino AcidsThere are 20 different types of Amino Acids• Eight essential amino acidsEight essential amino acids

Cannot be made by the body, must be obtained Cannot be made by the body, must be obtained through diet.through diet.

Amino acidsAmino acids

Structure of proteinsStructure of proteins

• Primary structure: order of the amino Primary structure: order of the amino acidsacids

Secondary structure- first level of Secondary structure- first level of foldingfolding• Pleated sheet.Pleated sheet.

This type of folding makes smooth proteins This type of folding makes smooth proteins such as smooth muscle and silk. And such as smooth muscle and silk. And puppies. puppies.

-Tim-Tim

Diagram of a pleated sheetDiagram of a pleated sheet

Pleated sheetPleated sheet

-Alpha helix-Alpha helix• This type folding creates proteins that This type folding creates proteins that

are very springy such as hair, tendons, are very springy such as hair, tendons, and ligaments.and ligaments.

• All secondary structure is held together All secondary structure is held together with hydrogen bonding that exists with hydrogen bonding that exists between the C=O and NH groups on the between the C=O and NH groups on the amino acids.amino acids.

Alpha helixAlpha helix

Alpha helixAlpha helix

Tertiary foldingTertiary folding This is the final layer of folding for most This is the final layer of folding for most

proteins. This type of folding is made proteins. This type of folding is made through the “R” groups on the amino acids. through the “R” groups on the amino acids.

Examples on the appearance of tertiary Examples on the appearance of tertiary proteinsproteins

Tertiary folding cont.Tertiary folding cont. Held together by these attraction Held together by these attraction

between R groups:between R groups:• Ionic attractionIonic attraction• Covalent bondsCovalent bonds• Non polar attraction (Van der Waal)Non polar attraction (Van der Waal)• Dipole attractionDipole attraction• Hydrogen bondingHydrogen bonding

Also Between C=O and H-O- Also Between C=O and H-O- attractions on the amino acidsattractions on the amino acids

• Here are some examples of the tertiary Here are some examples of the tertiary folding of a complete proteinfolding of a complete protein

Quadinary foldingQuadinary folding Has more than one poly-peptide Has more than one poly-peptide

chainchain Held together by same attractions as Held together by same attractions as

tertiary folding.tertiary folding. These proteins are often enzymes These proteins are often enzymes

and hormonesand hormones Tend to be globular in shapeTend to be globular in shape

example:Hemoglobin - Globular to fit example:Hemoglobin - Globular to fit Fe+Fe+33

Denaturing proteinsDenaturing proteins Denaturing is when a change is Denaturing is when a change is

made to the tertiary and quadinary made to the tertiary and quadinary structure of the proteins. Denaturing structure of the proteins. Denaturing can be achieved through changes in:can be achieved through changes in:• TemperatureTemperature• pH – pH – • Metal ions—interfere with ions and Metal ions—interfere with ions and

attractions between amino acidsattractions between amino acids

Identifying Amino AcidsIdentifying Amino Acids There are two tests commonly used in There are two tests commonly used in

identifying amino acids: chromatography identifying amino acids: chromatography and electrophoresis.and electrophoresis.

In both tests, proteins are broken down In both tests, proteins are broken down into fragments or into individual Amino into fragments or into individual Amino Acids first through the use of restriction Acids first through the use of restriction enzymes. Once the protein is in A.A. or enzymes. Once the protein is in A.A. or small fragment form, the following two small fragment form, the following two tests can be used.tests can be used.

ChromatographyChromatography Separates A.A. on differences of molecular Separates A.A. on differences of molecular

weight and solubility in solvent.weight and solubility in solvent. Chromatography strip is placed in a Chromatography strip is placed in a

solvent. A.A. are placed on the strip above solvent. A.A. are placed on the strip above the solvent levelthe solvent level

The strip absorbs the solvent. As the The strip absorbs the solvent. As the solvent travels up the strip the A. A. are solvent travels up the strip the A. A. are picked up and travel with it. The smaller picked up and travel with it. The smaller or more soluble A.A. will move faster.or more soluble A.A. will move faster.

Chromatography cont.Chromatography cont. When the solvent reaches the top of the strip, the When the solvent reaches the top of the strip, the

strip is removed from the solvent and strip is removed from the solvent and identification begins.identification begins.

The strip is sprayed with ninhydrin to make the The strip is sprayed with ninhydrin to make the A.A. show up.A.A. show up.

The distance each A.A. moves is measured.The distance each A.A. moves is measured. The distances are calculated as ratio: The distances are calculated as ratio: DistanceDistance

traveled by A.A./Distance traveled by solventtraveled by A.A./Distance traveled by solvent This ratio value, Called RThis ratio value, Called Rff, is then compared to a , is then compared to a

known Rknown Rf f that is listed on a table for each A.A. that is listed on a table for each A.A. Thus, an A.A. can be identified by its RThus, an A.A. can be identified by its Rff value. value.

ElectrophoresisElectrophoresis Separates A.A. on their differences in Separates A.A. on their differences in

molecular weight or their charge, or pH.molecular weight or their charge, or pH. Each R group has a different charge. This Each R group has a different charge. This

difference affects both the solubility and difference affects both the solubility and its migration rate in an electric field. (How its migration rate in an electric field. (How fast it moves when voltage is applied to it)fast it moves when voltage is applied to it)

General electrophoresis General electrophoresis A.A.s are placed in wells in a buffered gel A.A.s are placed in wells in a buffered gel

w/ a specific pH, and then voltage is run w/ a specific pH, and then voltage is run across the gel for a period of time.across the gel for a period of time.

The A.A.s will migrate at different ratesThe A.A.s will migrate at different rates Stop the voltage (note migration rates)Stop the voltage (note migration rates) A.A.s are identified based upon these rates A.A.s are identified based upon these rates

by matching the migration rate up with a by matching the migration rate up with a known migration rate for an amino acid.known migration rate for an amino acid.

Problems with electrophoresisProblems with electrophoresis The pH of the gel is crucial. Every A.A. has pH The pH of the gel is crucial. Every A.A. has pH

in which it will not migrate. This is called its in which it will not migrate. This is called its iso-electric point or pI. At this point the A.A. iso-electric point or pI. At this point the A.A. has both (+) and (-) in charge so no migration has both (+) and (-) in charge so no migration will occur. Methods of avoiding this problem:will occur. Methods of avoiding this problem:• avoid using a gel with a pH close to the pI point of avoid using a gel with a pH close to the pI point of

an A.A. or run several tests with gels of different an A.A. or run several tests with gels of different pHs. pHs.

• Use a gradient pH gel. That is, the pH gradually Use a gradient pH gel. That is, the pH gradually changes through out the gel. Each A.A. will migrate changes through out the gel. Each A.A. will migrate until it reaches its pI. This method is often used.until it reaches its pI. This method is often used.

pH of some Isoelectric pointspH of some Isoelectric points

Amino acidAmino acid

Glutamic acidGlutamic acid PhenylalaninePhenylalanine SerineSerine HistidineHistidine argininearginine

pH of isoelectric pH of isoelectric pointpoint

3.23.2 5.55.5 5.75.7 7.67.6 10.810.8

Another method Another method

Chemically treat the amino acids so Chemically treat the amino acids so that each one has the same charge. that each one has the same charge. This makes the amino acids migrate This makes the amino acids migrate on the basis or their size, that is, on the basis or their size, that is, molecular weight. Lighter acids molecular weight. Lighter acids migrate faster and so travel further migrate faster and so travel further in a given time frame. in a given time frame.

Electrophoresis chamberElectrophoresis chamber

Diagram of an electrophoresis gelDiagram of an electrophoresis gel

MIGRATE

Wells w/ A.A.

A gel with an electric field applied to it. The A.A. migrate across.

Photograph of an electrophoresis Photograph of an electrophoresis gel. The test was run 4 times.gel. The test was run 4 times.