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ProteinsProteins

The Function of ProteinsThe Function of Proteins

Amino AcidsAmino Acids

The Peptide BondThe Peptide Bond

Structure of ProteinsStructure of Proteins

Myoglobin, Hemoglobin and OxygenMyoglobin, Hemoglobin and Oxygen

Overview of Protein Structure and FunctionOverview of Protein Structure and Function

Effect of Temperature and pHEffect of Temperature and pH

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• Proteins are among the “essential” compounds necessary for the normal functioning of a living system.

• The name is derived from the Greek word “ProteiosProteios”, meaning firstfirst.

• All proteins are made from amino acids.

NH2 CR

HC

OH

O

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The function of proteinsThe function of proteinsEnzymesEnzymes Biological catalysts.

AntibodiesAntibodies They fight off infection. TransportTransport Move materials around

Ex. hemoglobin for O2.

RegulatoryRegulatory As hormones, they control metabolism.

StructuralStructural coverings and supportskin, tendons, hair, nails, bone.

MovementMovement muscles, cilia, flagella.

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Proteins are often gigantic in sizeProteins are often gigantic in size

– InsulinInsulin: Molecular Wt = 57005700

– HemoglobinHemoglobin:: Molecular Wt = 64,00064,000

– Virus ProteinsVirus Proteins: Molecular Wt = 40,000,00040,000,000

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Amino acidsAmino acidsAll these proteins are made from the same

building blocks.

• Twenty common amino acids.

• All are -amino-amino acids except proline.

• A primary amine is attached to the carbon.-carbon - the carbon just after the acid.

HH ||

R-C-COOHR-C-COOH || NHNH22

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Amino acidsAmino acids

Because an acid and base are both present, an amino acid can form a +/- ion, called a zwitterionzwitterion.

H H | |

R-C-COOH R-C-COO-

| | NH2 NH3

+

How well it happens is based on pH and the type of amino acid.

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-Amino acids-Amino acids

Except for glycine, the carbon is attached to four different groups - it is a chiral center.

For Carbohydrates We used the D-D- form.

For Amino AcidsWe use the L-L- form.

COO-

|H3N - C - H | R

COO-

|H3N - C - H | R

+

The amino group is on the left side of the fischerprojection.

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Classification of amino acidsClassification of amino acids

• The -amino acid group is the same in each of the amino acids.

• They are classified by the polarity of the side chain (R).HydrophobicHydrophobic - water fearing

non-polar side chainsHydrophilicHydrophilic - water loving

polar, neutral chains negatively charged positively charged

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Neutral, nonpolarNeutral, nonpolarside chainsside chains

HH33CC H

\\ |

HCHC-C-COO-

// |

HH33CC +NH3

valine

HH33CC H

\\ |

HC-CHHC-CH22-C-COO-

// |

HH33CC +NH3

leucine

H |

CHCH33--C-COO-

|

+NH3

alanine

H |

HH-C-COO-

|

+NH3

glycine

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Neutral, nonpolarNeutral, nonpolarside chainsside chains

H |

CHCH33 -S-CH -S-CH22-CH-CH22-C-COO-

|

+NH3

methionine

phenylalanine

H |

-CH-CH22-C-COO-

|

+NH3

proline HH22CC CH-COO-

|| |

HH22CC +NH2

HH22CC

HH33CC H

|| |

HH33C-CHC-CH22-CH-CH-C-COO-

|

+NH3

isoleucine

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Polar, neutral amino acidsPolar, neutral amino acids

tyrosine H |

-CH-CH22-C-COO-

|

+NH3

HO-HO-

H |

HO-CHHO-CH22-C-COO-

|

+NH3

serine HOHO H || |

CHCH33-CH-CH-C-COO-

|

+NH3

threonine

NN

H |

CHCH22-C-COO-

|

+NH3

tryptophan

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Polar, neutral amino acidsPolar, neutral amino acids

O O H |||| |

HH22N-C-CHN-C-CH22-CH-CH22-C-COO-

|

+NH3

glutamine

OO H |||| |

HH22N-C-CHN-C-CH22-C-COO-

|

+NH3

asparagine

H |

HS-CHHS-CH22-C-COO-

|

+NH3cysteine

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Acidic, polar side chainsAcidic, polar side chains

Based on having a pH of 7.

O O H |||| |-O-C-CHO-C-CH22-CH-CH22-C-COO-

|

+NH3

glutamic acid

OO H |||| |--O-C-CHO-C-CH22-C-COO-

|

+NH3

aspartic acid

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Basic, polar side chainsBasic, polar side chains

Based on a pH of 7. H

++ |

HH33N-CHN-CH22-CH-CH22-CH-CH22-CH-CH22-C-COO-

|

+NH3

lysine ++NHNH22 H

|| |

HH22N-C-N-CHN-C-N-CH22-CH-CH22-CH-CH22-C-COO-

|

+NH3

arginine H |

CHCH22-C-COO-

|

+NH3

histidine

NNNNHH HH

HH

+

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Essential Amino AcidsEssential Amino Acids

• Proteins are constantly being produced in the body for growth and repair.

• Of the 20 amino acids found in these proteins, 10 cannot be synthesized by the body.

• Arginine* Methionine• Histidine * Phenylalanine• Isoleucine Threonine• Leucine Tryptophan• Lysine Valine

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• Histidine is an essential amino acid for infants, but apparently not for adults.

• Arginine is produced in the body but not in sufficient quantities to meet protein demand.

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• Complete or Adequate ProteinsComplete or Adequate Proteins: supply all of the essential amino acids. (Animal Proteins)

• Incomplete ProteinsIncomplete Proteins: Low in one or more of the essential amino acids (Vegetable Proteins)

Animal ProteinsAnimal ProteinsSource Type of Protein Missing AAEgg Complete NoneMilk(Dairy)Complete NoneMeat, fish Complete None

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Vegetable ProteinsVegetable ProteinsSource Type of Protein Missing AAWheat Incomplete LysineCorn Incomplete Lysine &

TryptophanRice Incomplete LysineBeans Incomplete Methionine

TryptophanPeas Incomplete Methionine

Quinoa Complete Hemp Complete

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• A complete assortment of amino acids can be obtained from a vegetable dietvegetable diet by pairingpairing a vegetable protein missing one essential amino acid with a vegetable that contains it.

• The two vegetable proteins are called complementary proteins.

– Ex. Rice and Beans

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Amphoteric Properties of Amino Amphoteric Properties of Amino AcidsAcids

• Amphoteric substances act as acids acids or basesbases.– They are acids when they donate protons.– They are bases when they accept protons.

• Amino acids can act as acids or bases.act as acids or bases.–When placed in an acidic solution (low pH),

they act as bases by accepting protonsact as bases by accepting protons and becoming positively chargedpositively charged.

– In basic solutions (high pH), they act as act as acids by donating protonsacids by donating protons and becoming negatively chargednegatively charged.

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NH3 CH

CH3

C

O

O

+-

NH3 CH

CH3

C

O

OH

+ NH2 CH

CH3

C

O

O-

NET CHARGE + 1

NET CHARGE - 1

H+

AcidSolution

BasicSolution

OH-

ALANINE

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• Amino Acids function as buffersbuffers because they can neutralize small increases of acid or base.

• Proteins are one of the major bufferingmajor buffering systems in the body.

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ISOELECTRIC POINT (pI)ISOELECTRIC POINT (pI)

• A Zwitterion, which is electrically neutral overall, can only exist at a specific pHexist at a specific pH value.

• This pH value, called the isoelectric pointisoelectric point, is

different for each amino acid.• Amino acids with hydrocarbon R groups attain

their isoelectric point between pH 5.0 and 7.0

• ex. Leucine pH = 6.0• Basic amino acidsBasic amino acids need high pHhigh pH values to

reach their isoelectric points.• ex. Arginine pH = 10.8

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• Acidic aminoAcidic amino acids need low pHlow pH values.• ex. Aspartic acid pH = 3.0

• ProteinsProteins also have isoelectric pointsisoelectric points depending on the amino acids that make them up.– At their pH,At their pH, proteins become insolubleinsoluble in

water, clump together, and precipitateprecipitate out of solution.

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The peptide bondThe peptide bond

ProteinsProteins are polymerspolymers made up of amino acidsof amino acids.

Peptide bondPeptide bond - how the amino acids arelinkedlinked together to make a

protein.

HH ||

HH22NCNCCOCOOHOH

|| RR

++

HH ||

HH22NNCCOOHCCOOH

|| R’R’

H H OO | | ||||

HH22N - C - N - C - C -C -

|| RR

HH ||N N - C - COOH- C - COOH || | |HH R’ R’

This is a condensation reactioncondensation reaction: H2O is eliminated.

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• This bond between the two amino acids is called a peptide bondpeptide bond.

• Two amino acids joined like this give what is called a dipeptide.dipeptide.

These 2 amino acids could also link the other way.

NH2 CH

CH3

C

O

OH+ NH CH

H

C

O

OHH

NH2CH

CH3

C

O

NHCH

H

C

O

OH

Alanine Glycine alanylglycine (ala-gly)

-H2O

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• Any two amino acids can be joined in a similar manner to form dipeptides.

• It doesn’t end here !It doesn’t end here ! Each dipeptide still has a COOH and an NH2 that can form new peptide bonds.

• Adding a 3rd amino acid gives us a tripeptide.• This process can be continued to get a

tetrapeptide, a pentapeptide, and so on until we have a chain of hundreds or even thousands of amino acids.

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• The chains of amino acidschains of amino acids are the proteins.proteins.• The shorter chainsshorter chains are often called

polypeptidespolypeptides.– Ex. Glucagon with 21 amino acids21 amino acids is a large

polypeptidepolypeptide..– Insulin with 51 amino acids51 amino acids is a very small

proteinprotein..• We will consider a protein to be a peptide

chain with a minimum of 30 amino acids.minimum of 30 amino acids.

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Primary structurePrimary structureof proteinsof proteins

• Primary StructurePrimary Structure: What are the amino acids that make up the protein and how are they arranged in the chain ? (The amino acid The amino acid sequencesequence)

alaala argarg asnasn aspasp glnglncyscys gluglu

phephemetmetlyslysleuleuhishis ileileglygly

propro serser thrthr trptrp valvaltyrtyr

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• The amino acids in a chainamino acids in a chain are often referred to as residuesresidues.– Ex. Ala-gly-lys 3 residue amino acids3 residue amino acids

• The amino acid residue with the free COOHfree COOH group is called the C-terminalC-terminal,, and the amino acid residue with the free NHfree NH22 group is called the N-terminalN-terminal.

• Peptide and protein chains are always written with the N-terminalN-terminal residue on the residue on the leftleft.

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PeptidesPeptides

N-terminalresidue

N-terminalresidue

H H O O | | || ||

HH22N - C - C N - C - C

|| RR

HH ||N - C - COOHN - C - COOH | | | |H H R’’ R’’

H H OO | | || ||- NH - C - C -- NH - C - C - || R’R’

C-terminalresidue

C-terminalresidue

peptidelinkages

peptidelinkages

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• The continuingcontinuing pattern pattern of peptide linkagespeptide linkages is called the backbonebackbone of the protein molecule.

NHCH C

O

NHCH C NHCH

OR R' R"

C

O

The R groups are called the side chainsside chains.

The 20 different amino acid side chains provide variety and determine the chemicaland physical properties.

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• Each peptide and protein molecule in biological organisms has a different sequence of amino acids.

• It is this sequence that allows the protein to carry out its function, whatever it might be.

• The numbernumber of different protein possibilities is staggeringstaggering..– Ex. A tripeptide can have 20 different amino

acids at each position.• 20 x 20 x 20 = 8000 possible tripeptides8000 possible tripeptides

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• A typical protein with 60 amino acid residues can have up to 2060 different arrangements.

• This means that there would be 1 x 101 x 107878 possibilities.

• 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000, 000,000.

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Secondary structureSecondary structureof proteinsof proteins

Long chains of amino acids will commonly fold or curl into a regular repeating structure.

Structure is a result of hydrogen bonding between amino acids within the protein.

Common secondary structures are: - helix- helix - pleated sheet- pleated sheet

Secondary structure adds new properties to a protein like strength, flexibility, ...

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-Helix-Helix

One common type ofsecondary structure.

Properties of -helix include strength and low solubility in water.

Originally proposed byPauling and Corey in 1951.

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-Helix-Helix

H|N

C||O

H|N

C||O

H|N

C||O

C||OH

|N

C||O

H|N

C||O

H|N

C||O

C||O

C||O

H|N

H|N

H|N

Every amide hydrogen and carbonyl oxygen is involved in a hydrogen bond.There are 3.6 amino acids in each turn.

Multiple strands may entwine to make aprotofibrilprotofibril.

The R groups extend out fromthe helical portion of the -helix

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-Helix example-Helix example

myosin/actin structuremyosin/actin structureProteins used in muscle

actintroponin

myosin head myosin tail ATP and actinbinding sites

thick filament

thin filament

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-Pleated sheets-Pleated sheets

Another secondary structure for protein.Held together by hydrogen bonding between adjacent sheets of protein.

C|R

R|C

R|C

R|C

R|C

C|R

C|R

C|R

C|R

C|R

N|H

N|H

N|H

O||C

O||C

O||C

O||C

C||O

C||O

C||O

C||O

H|N

H|N

H|N

H|N

N|H

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-Pleated sheets-Pleated sheets

Silk fibroinSilk fibroin - main protein of silk is an example of a pleated sheet structure.

Composed primarily ofglycine and alanine.

Stack likecorrugatedcardboard for extra strength.

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Linus PaulingLinus Pauling

http://www.youtube.com/watch?v=yh9Cr5n21EE

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CollagenCollagen

Family of related proteins.

About one third of all protein in humans.

Structural proteinStructural proteinProvides strength to bones, tendon, skin, blood vessels.

Forms triple helix - tropocollagentropocollagen.

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•As an animal grows older, the extent of cross-linking increases and the meat gets tougher.

•Treatment with boiling water converts collagen to gelatin. Therefore, cooking meat converts part of the tough connective tissue to gelatin, making the meat more tender. (ex. Stewing chickens)

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Tanning hides increases the degree of cross-linking, converting skin to leather.

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• Wool, hair and muscle are all formed from strands of alpha helixes.

• These proteins can be stretched because the hydrogen bonds can be elongated and then return to the original configuration.

• This is especially true for wool.

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DISULFIDE BRIDGESDISULFIDE BRIDGES

• Disulfide bridges are covalent bonds formed when 2 cysteine units are oxidized to form a cystine unit.

SH

SH

oxidation

reduction

S

S

The strength of this bond is much greater than thatof a hydrogen bond.

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Fibrous proteinsFibrous proteins• insoluble in water• form used by connective tissues• silk, collagen, -keratins

Globular proteinsGlobular proteins• soluble in water• form used by cell proteins• 3-D structure - tertiarytertiary

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Tertiary structure of proteinsTertiary structure of proteins

• This refers to how the molecule is folded. It makes the molecule very compact.

• Results from interaction of side chainsside chains.

• Protein folds into a tertiarytertiary structure.

• This is typical of proteins called globular.globular.– Found in egg and serum albumin,

hemoglobin and myoglobin, and enzymes and antibodies.

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Types of tertiary bondingTypes of tertiary bonding

Possible side chain interactions:Possible side chain interactions:

- Similar solubilitiesSimilar solubilities

- Ionic attractionsIonic attractions

- Attraction between Attraction between ++ and and -- sidechains sidechains

- Covalent bondingCovalent bonding

Tertiary structureTertiary structureof proteinsof proteins

- S - S -

Salt bridge

SulfideCrosslink

Hydrogenbonding

Hydrophobicinteraction

-COO- H3N+-

-O \

H

-O \

H

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Side chain interactionsSide chain interactions Help maintain specific structure.

Oxidation of cysteine - crosslink formation. O ||

HO-C-CH-CH2-SH

|

NH2

O ||

HS-CH2-CH-C-OH

|

NH2 O ||

HO-C-CH-CH2-S -S -

|

NH2

O ||

SS-CH2-CH-C-OH +H2O

|

NH2

oxidation [O]

covalentdisulfide

bond

covalentdisulfide

bond

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Hydrophobic attractionsHydrophobic attractionsAttractions between R groups of non-polarnon-polar amino acids.

Hydrogen bondingHydrogen bondingInteraction between polarpolar amino acidR groupsR groups.

Ionic bondingIonic bondingBonding between oppositely chargedoppositely charged amino acid R groups.R groups.

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Quaternary structureQuaternary structureof proteinsof proteins

Many proteins are not single peptide strands.

They are combinationscombinations of several proteins- aggregate of smaller globular proteins.

Conjugated proteinConjugated protein - incorporate another type another type of groupof group that performs a specific function.

- prosthetic groupprosthetic group

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Quaternary structureQuaternary structureof proteinsof proteins

Aggregate structureAggregate structure

This example shows four different proteins and two prosthetictwo prosthetic groups.groups.

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Hemoglobin and myoglobinHemoglobin and myoglobin

HemoglobinHemoglobinoxygenoxygen transporttransport protein of red blood cellsred blood cells.

MyoglobinMyoglobinoxygen storage protein of skeletal muscles.skeletal muscles.

Both proteins rely on the heme groupheme group as the binding site for oxygen.

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MyoglobinMyoglobin

HemeHeme

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HemoglobinHemoglobin

4 heme

2 chains

2 chains

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Hemoglobin and Hemoglobin and oxygen transportoxygen transport

In the lungs, there is an abundance of OO22 so

oxygen is picked upoxygen is picked up by the hemoglobin.

When blood reaches the cells, there is a lack of O2 so oxygen is given upoxygen is given up by the hemoglobin.

Hb + 4 O2 Hb(O2)4

Hb + 4 O2 Hb(O2)4

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Sickle cell anemiaSickle cell anemia

Defective gene results in production of mutant mutant hemoglobinhemoglobin - one misplaced amino acid. Glutamate is replaced by valine at 2 of the 547 2 of the 547 positions.positions.Still transports oxygen but results in deformed blood cells - elongated, sickle shapedsickle shaped.Difficult to pass through capillaries. Causes organ damage, reduced circulation.

Affects 0.4 % of American blacks.

Sickled cellsSickled cells

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Summary ofSummary ofprotein structureprotein structure

primary secondary

tertiary quaternary

H H O O | | || ||

HH22N - C - C N - C - C

|| RR

HH ||N - C - COOHN - C - COOH | | | |H H R’’ R’’

H H O O | | || ||- NH - C - C -- NH - C - C - || R’R’

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Effect of temperatureEffect of temperature and pH on proteins and pH on proteins

• Both will alter the 3-D shapealter the 3-D shape of a protein if you go beyond a ‘normal’ range.

• Disorganized protein will no longer act as intended - denatureddenatured. They become biologically inactivebiologically inactive.

• They will clump together - coagulatecoagulate.– Examples - fryingfrying an egg an egg

HClHCl in stomach in stomach

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Denaturing of a proteinDenaturing of a protein

heator

acid

heator

acid

heator

acid

heator

acid

denatured coagulated

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•Changes in pH or temperature may not may not break any of the break any of the peptide bonds.peptide bonds.•The primary primary structure is maintained.maintained.•If denaturationdenaturation occurs under extremely mild mild conditionsconditions, the protein may be restored to its original shape.

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COAGULATION:COAGULATION:•Most changes are so drastic that the protein protein remains denaturedremains denatured.•Protein strands become insolublebecome insoluble and precipitate out of solution (coagulatecoagulate).•Effects of coagulation are irreversible.irreversible.

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HydrolysisHydrolysis

• Will result in proteinprotein being reduced to simpler peptides and amino acidspeptides and amino acids.

• Amount of hydrolysis depends on pH, time and pH, time and temperature.temperature.

OO ||||

HH22N - CH - C -N - CH - C -

|| RR

OO ||||

NH - CH - C - OH NH - CH - C - OH + H+ H22OO

|| R’R’

OO ||||

HH22N - CH - C - OHN - CH - C - OH

|| RR OO ||||

HH22N - CH - C - OHN - CH - C - OH

|| R’R’

H+ or

OH-

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• Proteins are often biologically activebiologically active only at certain pH ranges.–For most enzymes, the optimum pH is

7.0 to 7.57.0 to 7.5– Two exceptionsTwo exceptions are the digestive

enzymes pepsin and trypsinpepsin and trypsin.• PepsinPepsin in acidic stomach: pH = 2.0• TrypsinTrypsin in small intestine: pH = 8.0

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• The effect of acid or base is to add or removeadd or remove HH++ to ionic side groups.– Salt bridgesSalt bridges and hydrogen bondshydrogen bonds are

disrupted.• When a protein is placed in a strong acid or

base, coagulation may also occur.coagulation may also occur.– Ex. Cheese made from acid coagulated

protein (casein) which forms curds.

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• Tannic acidsTannic acids in burn ointments cause protein protein coagulationcoagulation at the site of the burn.

• This forms a protective coatingprotective coating which acts as a barrier to further loss of fluidsbarrier to further loss of fluids.

• A household source of tannic acid is Tea.Tea.– Applying dampened tea bagsApplying dampened tea bags to a burn will

precipitate protein and form a protective barrier over the wound.

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Heat and UV LightHeat and UV Light

• Optimum temperatureOptimum temperature for most proteins is 37C37C..

• Very few proteinsVery few proteins remain biologically active biologically active above 50 Cabove 50 C. (Some bacteria have protein that remains stable up to 70 C and higher)

– Increased Thermal activityIncreased Thermal activity (heat or UV) disruptsdisrupts some of the hydrogen bonds and hydrogen bonds and attractions between non-polar side groupsattractions between non-polar side groups that maintain secondary and tertiary structures.

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• When you cook foodcook food, you are denaturing denaturing protein.protein.– Ex. Boiling an egg, frying a steak.

• High temperatures are used to disinfect surgical instruments, gowns, and gloves.– AUTOCLAVEAUTOCLAVE..

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ORGANIC SOLVENTSORGANIC SOLVENTS

• Solvents like Ethanol, isopropyl alcohol, and acetone disrupt the hydrogen bonding of proteins by forming their own hydrogen bonds with the protein.

• These solvents are used as disinfectants.– A 70% solution of ethanol or IPA can pass

thru cell walls of bacteria– Once inside, they cause coagulation of the

bacterial proteins within the cell.• A 90% solution isn’t nearly as effective. Why?

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Heavy Metal IonsHeavy Metal Ions

• Metal ions like Ag+, Pb 2+, Hg 2+, etc.. cause denaturation of protein.

• The heavy metals react with the disulfide bonds and the carboxyl groups of acidic amino acids.

• The denatured protein is insoluble and precipitates out of solution.– Ex. 1% AgNO1% AgNO33 placed in eyes of newborn to

kill the bacteria that causes gonorrhea.gonorrhea.

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Other Methods of DenaturationOther Methods of Denaturation

• AgitationAgitation: Violent whipping action causes a stretching of globular proteins which turns egg whites into meringues and whipping creams into toppings. How does cream of tartar work?How does cream of tartar work?– Which would be best for beating eggs into Which would be best for beating eggs into

meringue: a glass bowl or a copper bowl?meringue: a glass bowl or a copper bowl?– Why can canned pineapple be used in Why can canned pineapple be used in

gelatin deserts while fresh pineapple can’t gelatin deserts while fresh pineapple can’t be used?be used?

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Protein hormone examplesProtein hormone examples

Source ActionGastrin Stomach Causes stomach to

secrete HCl

Glucagon Pancreas Stimulates liver to metabolize glycogen

Insulin Pancreas Control of glucose inblood

Prolactin Pituitary Stimulates lactation

Vasopressin Pituitary Decreases level of urineprodction

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Protein galleryProtein gallery

Human growth hormoneHuman growth hormone 596 residues

Originally obtainedfrom human cadavers.

It would cost $20,000per year to treat onechild.

Now produced bygenetically engineeredbacteria.

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Protein galleryProtein gallery

Immunoglobin FCImmunoglobin FC - 262 residualsA ‘Y” shaped protein actually composed of 4 protein chains linked by disulfide bonds.

antigen-bindingsite

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Protein galleryProtein gallery

LipoproteinLipoprotein116 residues 2 helical strands