1

N C

R1

C N C

R2

C

H

H

H

H

H

H

H HO

O

O

O

H2O

N C

R1

C

H

H

H

H

N C

R2

C

HO

O

O

H

peptide bond

Amino acids link together through covalent bonds to form proteins(18.7)

proteins are polymers of amino acids linked head to tailshort polymers of amino acids (short proteins) are called peptidespeptide bond - the covalent bond that links amino acids together carboxylic acid from one aa and amino group from anotherwater is releasedresidue - an individual amino acid in a protein

2

Peptides and proteins are directional

amino terminus - end with free amino groupcarboxyl terminus - end with free carboxylic acid groupproteins are synthesized and specified from the amino to carboxyl terminus

when writing peptide names using full aa names, drop the “ine” from each aa except thecarboxyl end and replace with “yl”

carboxyl terminusamino terminus

N C

R1

C

H

H

H

H

N C

R2

C

HO

O

O

H

N C

R2

C

H

H

H

H

N C

R1

C

HO

O

O

H

R1R2

R2R1

N C

R1

C

H

H

H

H

N C

R2

C

HO

H

O

N C

R3

C

H

H O

N C

R4

C

H

H O

O

3

Threonine and valine can combine to form two different dipeptides. Draw them and writetheir names using full names, three letter and one letter abbreviations.

4

Identify the amino acids in the following dipeptide and tripeptide, and write their namesusing full names, three letter and one letter abbreviations.

5

The peptide bond has partial double bond character

you cannot rotate around the C=O or C-N bonds.causes the peptide bond and protein backbone to be planar (flat)side chains alternate above and below plane

The peptide bond is trans

the carboxyl oxygen and amide hydrogen are on opposite sides to minimize stericinteractions of the oxygen and hydrogen (overlapping of e- clouds = repulsion)

N C

R1

C

H

H

H

H

N C

R2

C

HO

O

O

H

the electrons in the double bond here, are shared a lot with this bond (the C-N bond)

6

Four Tiers of Protein Structure (18.8-18.11) Primary structure (1° structure)amino acid sequence of the protein written from amino to carboxyl terminusThe amino acid sequence determines its 3D structure (which then determines its properties.)depends solely on covalent bondsWhy determine primary structure?1. Determining primary structure is the first step in characterizing a protein - can help determine the function of a protein by comparison to other protein sequences2. primary structure determines 3D structure (which then determines its properties.)3. changes in 1° can make protein fold differently or cause disease (sickle cell anemia results from a change of 1 amino acid in hemoglobin glu to val)

1° Structure determines 3D shape and therefore protein function (or dysfunction)

7

Experimental determination of 1° structure

STEP 5

STEP 6

8

Experimental determination of 1° structureSplit the protein into several tubesStep 1 - use for amino acid constituent analysisDetermine which amino acids are present and in what proportionsuse high acid concentration and high temps (100-110 C) for 12-36 hourshydrolyzes peptide bondsdetermines number of possible cleavage sites for steps 3 and 4.resulting solution put through an amino acid analyzer to aa present and ratiosonly identifies which amino acids are present and ratios- not their orderStep 2 - use for end group analysisIdentify N-terminal and C-terminal amino acidscan be used to determine the number of polypeptide chains - process is automatedN-terminalEdman degradation

reliable forpolypeptidechains upto 40-60amino acids

+

N C SPhenyl isothiocyanate(Edman’s reagent)

HN C

S

NH 1 2 3 4 5 n COO-

1 2 3 4 5 nH3N COO-

Original peptide

Modified peptide

2 3 4 5 nH3N+

COO-+N

CNH

CHC

O

R1

S

amino acidderivativecan be easilyidentified

Can be used in anotherround as in steps 3 and 4

9

Step 2 (cont.)C-terminalEnzymatic analysis with carboxypeptidases

carboxypeptidases are exopeptidase (cleave end bonds)Four carboxypeptidases are used since not one enzyme will use each aa as substrate

Carboxy means itworks on thecarboxyl endof the protein

Peptid becausethey cleave

peptide bonds

“ase” is theending givento denote an

enzyme

1 2 3 4 5H3N+

COO-

Original peptide

n-1 n

Treat with carboxypeptidases H2O

1 2 3 4 5H3N+

COO-n-1

+

H3N CH

Rn

COOamino acid is easily identified

COO-n

10

Steps 3 and 4- Fragmentation of the Polypeptide Chaingoal is to produce the correct size polypeptide fragments for sequencing through

Edman degradation (Step 4 below)automated Edman is reliable for polypeptide chains up to 40-60 amino acidsmost proteins are >100 aa so the protein must be broken into fragmentsbreak up large protein into smaller polypeptides using endopeptidases & cyanogen bromideSite Specific peptidases - enzymes that cleave peptide bonds at very specific sites in

the proteinMost commonly used endopeptidases:Enzyme name Peptide bond cleavage SpecificityTrypsin cleaves after R or K amino acidsChymotrypsin cleaves after Y, W, and F

Commonly used chemical:cyanogen bromide (CN-Br) - cleaves after Mwill see all fragments end in M except 1 and that is the carboxyl end of the protein

N-ala-ser-phe-pro-lys-gly-gly-met-arg-trp-asp-met-gly-tyr-lys-ala-cys-C

chymotrypsin

trypsinCN-Br

11

ala-ser-phe-pro-lys-gly-gly-met-arg-trp-asp-met-gly-tyr-lys-ala-cys

chymotrypsin

trypsinCN-Br

Chymotrypsin digestion yields 4 fragments (remember, their sequences are unknowon at this time):

ala-ser-phepro-lys-gly-gly-met-arg-trp

asp-met-gly-tyrlys-ala-cys

Trypsin digestion yields 4 fragments also:

ala-ser-phe-pro-lysgly-gly-met-arg

trp-asp-met-gly-tyr-lysala-cys

Cyanogen bromide digestion yields 3 fragments:

ala-ser-phe-pro-lys-gly-gly-metarg-trp-asp-met

gly-tyr-lys-ala-cys

12

What fragments are produced by trypsin, chymotrypsin, and CNBr digestion of the following peptides?

1) gly-met-lys-gly-ala-cys-met-asp-trp-arg-met-val-tyr-iso-ala-cys-met-phe-leu

1) VGCMAWGYLEMTSRGGF

Trypsin fragmentsgly-met-lys gly-ala-cys-met-asp-trp-arg met-val-tyr-iso-ala-cys-met-phe-leu

Chymotrypsin fragmentsgly-met-lys-gly-ala-cys-met-asp-trp arg-met-val-tyr iso-ala-cys-met-phe leu

CNBr fragmentsgly-met lys-gly-ala-cys-met asp-trp-arg-met val-tyr-iso-ala-cys-met phe-leu

Trypsin fragmentsVGCMAWGYLEMTSR GGF

Chymotrypsin fragmentsVGCMAW GY LEMTSRGGF

CNBr fragmentsVGCM SWGYLEM TSRGGF

13

Step 5- Sequence determination of each polypeptide from steps 3 and 4step 4 produces polypeptides whose sequences are still unknown. So, each polypeptideproduced from digestion with trypsin, chymotrypsin, or cyanogen bromide treatmentundergoes Edman degradation to determine its sequence.

Step 6 - Fragment alignmentThe sequences of the fragments determined in step 5 are put together like a puzzle toproduce the sequence of the original protein.

Example:A solution of a small protein of unknown sequence was divided into two samples. Onesample was treated with trypsin and the other with chymotrypsin. The smaller peptidesobtained by trypsin treatment had the following sequences:

Leu-Ser-Tyr-Ala-Ile-ArgAsp-Gly-Met-Phe-Val-Lys

The smaller peptides obtained by chymotrypsin treatment had the following sequences:

Val-Lys-Leu-Ser-TyrAla-Ile-ArgAsp-Gly-Met-Phe

Deduce the sequence of the original protein.Trypsin treatment indicates 2 basic aa in the peptide, one of which must be the c-terminal aa - otherwise another fragment would have been produced that lacked a basic residue at the c end - like in chymotrypsin, there is a piece that does not have an aromatic at the end.

14

Secondary structure (2° structure)common structures in proteins that result from H- bonding of the backbone atoms only

(the carbonyl and amide nitrogen)

The -Helix (alpha-helix) polypeptide backbone coils around an imaginary pole (like a telephone cord)

in the figure on the left, green lines denote hydrogen bonds betweenthe oxygen of C=O and the hydrogen of N-H

In this figure, the side chains of the amino acidsare shown in green and purple. Note how theystick out of the helix perpendicular to thedirection of the helix.

Most common 2° structure found in proteins.If you combine all of the amino acids fromall proteins whose structures are known,1/3 of the aa are found in an helix

15

Some proteins are composed entirely of -helices

There are 7 -helices in this polypeptide.This polypeptide is part of hemoglobin.

Hemoglobin has four of these polypeptides(called “subunits”) that interact non-covalently.

16

H-bonds are between the oxygen of a carbonyl and the amide hydrogen 4 amino acids away

What holds an alpha-helix together?Hydrogen bonds between carbonyl and amide nitrogen four aa away

hydrogen bonds shown by dotted lines

17

The -Sheet (beta-sheet) polypeptide backbone is stretched out like an extended accordion

There are two kinds of -sheets

parallel anti-parallel

18

What holds a beta-sheet together?Hydrogen bonds between carbonyl and amide nitrogen

H-bonds are between the oxygen of a carbonyl and the amide hydrogen on another strand

Strand 1 Strand

2

Strand 3 Strand

4

Strand 5

Antiparallel -sheet with 5 strands

19

Some proteins are composed almost entirely of -sheets

Retinol binding protein(pdb code 1ggl)

neuraminidase from influenza virus(pdb code 1EUU)

20

Some proteins have both -helices and -sheets

Carboxypeptidase (digestion protease)(pdb code 5CPA)

Hexokinase (glycolysis)(pdb code 5CPA)

21

Tertiary structure (3° structure)overall 3D shape of all of the atoms in the protein (not just those involved in

-helices or -sheets)proteins have different 3D shape compared to each other (though same family aresimilar) but each carboxypeptidase folds the same as every other carboxypeptidase

secondary structure - interactions of backbone atomstertiary structure - mainly results from interaction of side chains far apart in

primary sequence or side chain-backbone interactions - residues far apart inprimary sequence can be close together in space

hydrophobic residues usually buried interior and hydrophilic on exteriorShape determining interactions in proteins1. Hydrophobic interactions between non-polar side chains

2. H-bonds between side chains

3. H-bonds between backbone atoms

4. H-bonds between a side chain and a backbone atom

5. Salt bridge (ionic attraction between charged side chains)

6. Disulfide bonds (covalent bond between cysteine residues)

22

Shape-Determining Interactions in Proteins (18.8)

23

Quaternary structure (4° structure)some proteins have more than one amino acid chain (called subunits) that interact

non-covalently. The arrangement of these subunits with each other is 4° structure.

dimer - protein with 2 subunits (2 non-covalently linked polypeptide chains)trimer - protein with 3 subunitstetramer - protein with 4 subunits

Examples: Hemoglobin - tetramer ATP Synthase - trimer

24

Sickle Cell DiseasesGroup of diseases affecting shape and oxygen carryingcapacity of red blood cells.

mainly affects persons of African or Mediterranean descent (Italian, Spanish)

Inherited diseases - not contagiousDiseases are diseases of the protein hemoglobin

result from mutations in hemoglobin genes or lack of production of hemoglobin

autosomal recessive diseases

Disease and Protein Structure

oxygen binds here

Hemealphachain(141 aa)

alphachain (141 aa)

betachain (146 aa)

betachain(146 aa)

Hemoglobin is a tetramer

25

Hemoglobin genes

two sets of hemoglobin genes - one set from mother and one set from fathereach set contains 2 alpha genes (chromosome 16) and 1 beta gene (chromo 11)gives a total of 4 alpha genes and 2 beta genesin normal hemoglobin production, the amt. of alpha produced=amt of beta

alpha and beta chains have only 20% sequence homology but virtually identical 3DTypes of hemoglobin:

Hemoglobin A - normal hemoglobin - normal alpha and beta chains

Hemoglobin F - fetal hemoglobin - has higher affinity for oxy than Hgb A - production declines by 6 months of age- normal alpha chains; beta replaced by gamma chains

Hemoglobin S - normal alpha chains, mutated beta chain where Glu-6 is replaced by Val - sickle cell

Hemoglobin C genes - normal alpha genes, mutated beta genes where Glu-6 is replaced by lys

beta-thalassemia - normal genes but less beta produced

alpha - thalassemia - normal genes but less alpha produced

26

Sickle Cell Disease is notnecessarily Sickle Cell Anemia

Common Types of Hemoglobin proteins 1) Hemoglobin AA - normal, healthy hemoglobin

2) Sickle Cell Trait (AS) - alpha genes normal - one normal beta gene, one beta sickle- healthy and will have no symptoms associated with sickle cell disease. - both normal and sickle hemoglobin produced; mostly normal since binds to better

3) Sickle Cell Anemia (SS) - alpha genes normal - two sickle beta genes 4) Sickle C Disease (SC) - alpha genes normal - one sickle beta gene - one hemo C disease

5) Sickle beta-thalassemia (S/-thalassemia) - alpha genes normal - one sickle and thala

AA AA AS AS

AA AS AS AS

AAAA AS AS SS

Sickle Cell Trait PedigreeSickle Cell Anemia Pedigree

27

Sickle hemoglobin has same affinity for oxygen as normal hemoglobin but in low oxygen

areas (veins and venous capillaries), hemoglobin molecules clump togetherclumping causes together causing red blood cells to become sickle in shape and become hardsickle, hard red blood cells don’t flow through capillaries as easily - get caught and

block capillaries blocking blood and oxygen flow to tissuesshortness of breath, stokes, fatigue, infections, jaundice, lung blockage, leg ulcers,

bone damage, kidney damage, eye damage, and obviously low rbc counts. Normal rbc survive about 120 days but sickle rbc usually last 20 days - sickle cell patients have less rbc and so less hemoglobin

sickle red blood cellsnormal red blood cells

28

Hemoglobin Clumping in Sickle Cell Anemia:

Hemoglobin molecules clump under low oxygen concentrations (venous blood) due tohydrophobic interactions between different hemoglobin molecules

Hbg S - Glutamic acid at position 6 of thebeta chains are mutated to valine

- Hydrophobic patch on the beta chainof another hemoglobin molecule

- val-6 and hydrophobic patch (ala, phe, leu)interact

29

Diagnosing Sickle Cell Disease

Electrophoresis - uses a gel to separate molecules based on size, charge and/or shape

- At the pI of a specific protein - the protein molecule carries no net charge and does not migrate in an electric field. - At pH above the pI - the protein has a net negative charge and migrates towards the anode (the positive end). - At pH below the pI - the protein obtains a net positive charge on its surface and migrates towards the cathode (the negative end).

For proteins of same size, migration depends on net charge- the more negatively charged, the faster the migration towards anodecathode

anode

30

Hemoglobin electrophoresis

Test that determines the types of hemoglobin in a patient

different forms of hemoglobin will migrate in an electric field differently because of different charges:

Hemoglobin AHemoglobin S - changes glutamic acid (neg) to valine (neutral) - one less negative than A per beta chain so 2 less neg totalHemoglobin C - changes glutamic acid (neg) to lysine (pos) - two less negative than A and one less than S per chain - so, four less than A and two less than S

Cathode(-)

Anode(+)

Power Supply

Hbg A

Hbg S

Hbg C

origin

31

Determine the type of hemoglobin disease (if present) in patientswhose hemoglobin electrophoresis patterns are as follows:

origin

C

S

F

AC

ontr

ol

Pati

ent

1

Contr

ol

Pati

ent

2

Pati

ent

3

Pati

ent

4

Pati

ent

5

Pati

ent

6

+

- Patient Disease1

2

3

4

5

6

32

Disease and Protein Structure (Application on pg. 533)

Prions - “proteinaceous infectious particles”In 1997, Stanley Prusiner (a neurologist) won the Nobel Prize for Physiology

andMedicine for discovering prions

prions are naked proteins that infect and destroy brain tissuefirst time something other than organisms containing nucleic acid could

replicate andcause disease - met w/ high level of criticism - eventually most accepted

hypothesis

Spongiform Encephalopathy - general category of diseases caused by prions

Brains infected with prions look like a sponge. The holes are areas of brain cells that have died as a result of prion infection.

Greek for brain Greek for diseaseTakes the formof a sponge

Symptoms:dementia, Involuntary and irregular jerking movements,loss of vision, speech, coordination, depression,withdrawal

33

Prion diseases affect many speciescan be inherited or acquired:

Prion diseases can be a genetic disorder or can be acquired through:

1) Eating infected animal parts (brain/spinal cord) - cross species2) Brain surgery - prions remain infectious on surgical instruments after sterilization3) Corneal transplant 4) Contaminated growth hormone from pituitary glands5) spontaneous mutation

Prion Disease Other Names Species Infected

Bovine Spongiform Encephalopathy Mad Cow Disease or BSE Cows

variant Creutzfeldt-Jakob Disease nvCJD or vCJD acquired; humans from eating cattle with BSE

Kuru infectious; cannabilistic humans in Papua, New Guinea

Scrapie Sheep

Feline Spongiform Encephalopathy FSE Cats

Transmissible Mink Encephalopathy TME Mink

Chronic Wasting Disease Elk/Deer

Gerstmann-Sträussler-Scheinker disease GSS inherited disease of humans

Fatal Familial Insomnia FFI inherited disease of humans

Creutzfeldt-Jakob Disease CJD inherited disease of humans

34

Prions are Molecular TransformersKey players:

PrPc - protein normally attached outside nerve cells in brain - mainly -helical (“c” for cellular)Prpsc - identical in sequence to Prpc but is - mainly -sheet (“sc” for scrapie) - over-abundant in brain tissue of scrapie sheep

Prion theory: Prpsc has identical primary structure to Prpc but has a different 3D shape. Prpsc causes Prpc to change shape into more Prpsc which clog cells because normal cellular enzymes that destroy proteins are ineffective against Prpsc. Prpsc eventually causes infected cells to lyse which releases Prpsc into adjacent brain tissue to recruit even more Prpc to change shape - cycle continues.

PrPc PrPsc

Nothing added - nothingdeleted - just a change inshape!

35

Evidence that Prions are Proteins

When PrPsc is purified, scrapie infectivity is also purified

The amount of PrPsc directly coincides with infectivity levels

Procedures known to destroy nucleic acids do not destroy prion infectivity

PrP gene mutations that lead to inherited prion diseasealso produce PrPsc

Over-production of PrP increases rate of prion disease

PrP knock-out mice did not get prion disease after inoculation with prion material

36

Destroying Protein Structure (Section 18.12)

Protein denaturationProtein hydrolysis - hydrolysis of peptide bonds to produce amino acids

destroys all levels of protein structure - even primaryProtein denaturation - protein unfolding - primary structure still intact

primary structure intact

Review:What forces hold proteins in a specific 3-D shape?

Hydrogen bonding

van der Waals

salt bridges

hydrophobic

dipole-dipole

37

Methods of protein denaturation

pH changes - cause amino acids to alter their protonation state so can destroy hydrogen bonding and salt bridges

ionic concentration - high salt out-competes amino acids for each other

temperature - increased thermal motions break energy barriers - supplies enough energy to overcome the forces holding a molecule together

mechanical agitation - beating of egg whites causes proteins to denature

detergents - makes solution more hydrophobic so protein unfolds

organic compounds - polar solvents like acetone and ethanol - make solution more hydrophobic and also compete for hydrogen bonding with amino acid side chains

reducing/oxidizing agents - break/form disulfide bonds

most denaturation is reversible but there are many cases where the protein can renature when the reason for its denaturation is removed.