Module 2 - Topic 1 - Protein Structure & Function Cont.

7/30/2019 Module 2 - Topic 1 - Protein Structure & Function Cont.

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Summary of last lecture (recorded)

Amino acids are joined by peptide bonds

Differences in protein function result fromdifferences in amino acid composition and

sequence.

Secondary structure: alpha-helix and beta-sheets

Wide variety of 3D structures formed by folding

of various sections of secondary structure.

Proteins consist of Domains


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Conus victoriae Courtesy of Bruce Livett & David Paul

http://pubs.acs.org/cen/science/88/8830sci2.html

Tying the ends of a protein together makes the molecule resistant to

exopeptidase enzymes that chomp away at peptide termini. But Craik saysthe cyclization also enhances hydrogen bonding across the entire molecule,

making it resistant to the endopeptidases that attack a proteins interior amino

acids. He says its sort of like a zipper: A zipper can be regarded as a series

of hydrogen bonds all interlocking together, and when you zip it all up, youve

got a beautiful set of coordinated hydrogen bonds. But youve still got two

ends, and when you pull apart those two ends of the zipper, then the first

hydrogen bond goes, then the next, and then the next.


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The Engineering of an Orally Active Conotoxin for

the Treatment of Neuropathic PainClark R et al, Angewandte Chemie, 2010.


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-helices are stabilised by:1. Adjacent negatively

charged amino acids

2. Glycine residues

3. Large bulky amino acidsclose together

4. Hydrogen bonding


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In the -helix the hydrogen bonds:

1. are roughly parallel to the

axis of the helix.

2. are roughly perpendicular tothe axis of the helix.

3. occur mainly between

electronegative atoms of theR groups.

4. occur only between some of

the amino acids of the helix.

5. occur only near the amino

and carboxyl termini of the

helix.


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Thr and/or Leu residues tend to disrupt an a-helix when

they occur next to each other in a protein because:

1. an amino acids like Thr is highly

hydrophobic.2. covalent interactions may occur

between the Thr side chains.

3. electrostatic repulsion occurs

between the Thr side chains.

4. steric hindrance occurs between the

bulky Thr side chains.

5. the R group of Thr can form ahydrogen bond.


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-sheets are held in a side by side arrangement

mainly by:

1. disulfide bonds

2. H-bonding

3. interactions between

charged side chains.

4. lysine cross-links

5. none of the above


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Proline residues are usually present in:

1. -sheets

2. -sheets

3. -turns

4. -helices


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Tertiary structure 2 major groups of proteins

Fibrous with polypeptide chains in long chains or sheets

Globular with polypeptides folded into more or less spherical shape


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1. Keratins (-helical proteins)

2. Fibroin (-conformation)

3. Collagen (triple helix)4. Elastin

These proteins give strength and flexibility to the structures in which they occur.

Fundamental structure is simple repeating element of secondary structure.


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Major protein components of hair and fingernails as wellas comprising a major part of animal skin.

-keratins are part of a broader family of proteins called

intermediate filament proteins that play importantstructural roles in the cell cytoskeleton.

Consist entirely of-helices.

The -helices are cross-linkedby disulfide bonds.

http://www.immediart.com/catalog/images/bigger_images/SPL_6

_P780110-Fibroblast_cells_showing_cytoskeleton.jpg


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Hair consists of fibrils, which consist of even smaller fibres

twisted around each other.

Each fibre consists of 2 -helical polypeptides oriented in

parallel) coiled around each other to form a super-twisted coiledcoil. The super-twisting amplifies the strength of the overall

structure.

www.imb-

jena.de/.../proteins_classification.html


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The helical path of the super-twist is left-handed, the opposite

to the individual -helices.

The surfaces where the two -helices touch are made up ofhydrophobic residues with their R groups meshed together in a

regular interlocking pattern.

This permits close packing of the polypeptide chains within theleft-handed supertwist.

-keratin is rich in the hydrophobic residues Ala, Val, Leu, Ile,

Met and Phe.


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About 4 protofibrils 32 strands of-keratin combine to form an intermediate filament. A hair

is an array of many -keratin filaments.


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The intertwining of the two -helical polypeptides is an example

of quaternary structure.

The quaternary structure of-keratin can be quite complex with

many coiled coils assembled into large supramolecular

complexes (eg: filament of hair).

In -keratin the cross-links stabilizing the quaternary structure

are disulfide bonds.


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Bend hair around

an appropriate shape

Moist heat

breaks hydrogen bondsOxidizing agent makes

new disulfide bonds


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-keratins are stringy, insoluble proteins.

Autumn and Peattie (2002) Integrative Comp. Biol. 42, 1081

Beta-keratins form the hard corneous material of reptilian scales

Toni et al, J. Proteome Res., 2007, 6 (9), pp 33773392


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Fibroin, the component of silk and spider webs.

Consist of mostly -pleated sheet.

Rich in small amino acid residues such as glycine andalanine permitting close packing of-sheets and aninterlocking arrangement of R groups.

The overall structure is stabilized by extensive hydrogenbonding between all peptide linkages in the peptides ofeach -sheet and by optimization of van der Waalsinteractions between sheets.


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Composition of silk fibroin is 36% gly, 24% ala, 12 % ser(ratio 3:2:1) consisting of repeats of (gly-ser-gly-ala-gly-ala)n along the lengths of the polypeptides.

The -sheets are antiparallel containing small aminoacids packed tightly together and stabilized throughextensive hydrogen bonding to form strong inextensiblestrands.

hmaloy.wikispaces.com


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Silk does not stretch because it is already highly extended.However, the structure is flexible because it is held together by numerous

weak interactions rather than the by covalent bonds such as disulfide bonds.

http://en.wikipedia.org/wiki/Spider_silk Courtesy of University of Cambridge, UK

Department of Materials Science

Silk is remarkably strong, with a tensile strength similar to high grade steel.


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The fibers of silk and spider web are made up of the protein

fibroin, which consists of layers of anti-parallel sheets rich

in Ala and Gly residues.


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Is the most abundant protein in most vertebratesproviding the strength to connective tissues such astendons, cartilage, the organic matrix of bone and is animportant constituent of the skin.

The collagen helix is unique and quite distinct from an-helix.

It is a left-handed helix consisting of 3 residues per turnCollagen is also a coiled coil with distinct tertiary andquaternary structures.

Three chains are twisted around each other and thissuperhelical twist is right-handed.


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Left-handed helix with 3 residues per turn.

The sequence has a glycine residue in every 3rd positionand a high percentage of proline and hydroxyproline.

Every 3rd residue of the helix lies in the centre of thetriple helix. Due to size constraints this must be aglycine.

The amino acid sequence of collagen is generally a repeatof gly-X-Y where X is often pro and Y is oftenhydroxyproline.

Pro and hydroxyproline permit the sharp twisting of thehelix.


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The tight wrapping of the alpha

chains in the collagen triple helix

provides tensile strength greater thanthat of steel wire of equal cross

section.


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Tropocollagen is the basic unit of the collagen structure.

Hydrogen bonding between the 3 chains stabilises thetropocollagen triple helix.

Hydroxypro provide more avenues for hydrogen bonding.

Tropocollagen molecules pack side by side in anoverlapping fashion and are cross-linked to each other

via lysine residues to form a collagen fibre. This gives thestructure incredible strength.


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Fig. 4-11.Glycine is shown in red. Because of

its small size is required at the tight

junction where the three chains are in

contact.


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The tight wrapping of the of the chains in the collagentriple helix provides tensile strength greater then that ofsteel wire of equal cross section.

Collagen fibrils are supramolecular assemblies consistingof the triple-helical collagen molecules associated toprovide different degrees of strength.

Tropocollagen molecules pack side by side in anoverlapping fashion and are cross-linked to each othervia lysine, 5-hydroxylysine and histidine residues that arepresent at a few of the X and Y positions in collagen.

The rigid and brittle nature of aging is due to anaccumulation of covalent cross-links.


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Some human genetic defects illustrate the close relationshipbetween amino acid sequence and 3-dimensional structure.

Osteogenesis imperfecta (abnormal bone formation in babies)

and Ehlers-Danlos syndrome (loose joints) are both conditions

that can be lethal and both result from the substitution of an

amino acid with a larger R group (such as Cys or Ser or Arg)

for a single Gly residue in each chain

These single substitutions have catastrophic effect on collagen

function because they disrupt the Gly-X-Y repeat that gives

collagen its unique structure.

Ehlers Danlos syndrome (EDS)


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decrease in the tensile strength and integrity of the skin, joints,and other connective tissues.

Ehlers-Danlos syndrome (EDS)

"The India Rubber Man, The Elastic Lady," and "The Human Pretzel."

Family of diseases caused by abnormalities in the synthesis and

metabolism of collagen


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Epidermolysis Bullosa Keratins and Collagens (type VII)and others

Epidermolytic Hyperkeratosis Keratins

ebworld.faithweb.com/clinicalpics_j.htm


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Found in tissues such as ligaments arterial blood vesselswhich require highly elastic fibres.

The polypeptide of elastin is rich in glycine, alanine and

valine, and is very flexible and easily extended.Conformation is random coil which has little or nosecondary structure.

The polypeptide also contains frequent lysine residueswhich cross-link polypeptide chains.

The cross-links prevent the elastin fibres from extending

indefinitely, allowing them to snap back when the tensionis relieved.


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contains mostly Gly, Ala and Val with some Lys and Pro

www.imb-jena.de/.../proteins_classification.html


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The acid-base behaviour of a peptide can be

predicted from its free a-amino and a-carboxylgroups as well as the nature and number of its

ionizable R groups.

Like free amino acids, peptides have characteristic

titration curves and isoelectric points pI pH at which

they carry no nett charge, and do not move in an

electric field).

When the [H+] changes from 10-3 M


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When the [H+] changes from 10-3 M

to 10-5 M, the pH ..

1. Increases by 2 units

2. Decreases by 2 units

3. Stays the same

4. Increases 100 x

5. Decreases 100 x

T i h ldi 999 L 1 0 L f


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To a container holding 999mL water, 1.0 mL of

0.00001M HCl is added, what is the final pH?

1. 5

2. 7

3. 9

4. 3

T t i h ldi 999 L t 1 0 L f


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To a container holding 999mL water, 1.0 mL of

0.001M NaOH is added, what is the final pH?

1. 6

2. 7

3. 8

4. 105. 11

At pH 7 0 alanine has a net charge


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At pH 7.0 alanine has a net charge

of ..

1. +1

2. 0

3. -1


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An amino acid that has a charged side group at pH


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An amino acid that has a charged side group at pH

13 is:

1. leucine2. Lysine

3. Glutamine

4. Aspartic acid

5. Glycine

What percentage of a weak acid is ionized at a pH


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What percentage of a weak acid is ionized at a pH

value equal to its pKa?

1. 10%2. 25%

3. 50%

4. 75%

5. 90%

The pKa of the histidine side chain is 6.0. What is


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The pKa of the histidine side chain is 6.0. What is

the ratio of the acid form to the conjugate baseform at a pH of 8? ...

1. 0.01

2. 0.1

3. 1

4. 10

5. 100


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Consider the peptide AEILKVG

What is the net charge at pH 3.0, pH 8.0 & pH 12 ?


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Consider the peptide AEILKVGWhat is the net charge at pH 3.0, pH 8.0 & pH 12 ?

NH3

+-A-E-I-L-K-V-G-COOH

1. Remember that every peptide has an N-terminus and aC-terminus.


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Consider the peptide AEILKVGWhat is the net charge at pH 3.0, pH

8.0 & pH 12 ?

NH3+-A-E-I-L-K-V-G-COOH

2. Identify any amino acids that havea side chain that can be ionised,such as side chains with positivelyand negatively groups. In the caseof the above peptide this is Lysine

(K) and Glutamic acid (E).


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NH3

+-A-E-I-L-K-V-G-COOHpKa 9.7 4.3 10.5 2.3

3. Consider the pKa of each of the ionisable groups.

pH > pKa the proton tends to be offpH < pKa the proton tends to be on.pH = pKa half of the molecules are protonated and

half are deprotonated.


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NH3

+-A-E-I-L-K-V-G-COOHpKa 9.7 4.3 10.5 2.3 Net charge

+1 0 +1 -1 +1

pH > pKa the proton tends to be off.pH < pKa the proton tends to be on.pH = pKa half of the molecules are protonated and

half are deprotonated.


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NH3

+-A-E-I-L-K-V-G-COOHpKa 9.7 4.3 10.5 2.3 Net charge

+1 0 +1 -1 +1

+1 -1 +1 -1 00 -1 0 -1 -2

pH > pKa the proton tends to be off.pH < pKa the proton tends to be on.pH = pKa half of the molecules are protonated andhalf are deprotonated.

What is the net charge of the peptide


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g p p

Val-Gly-Asn-Ala at neutral pH (pH 7.0)?

1. + 12. 0

3. - 1

4. Unable to determinethe charge

5. Havent got a clue

Consider the peptide ALRKRG. What is the charge


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p p g

on the peptide a pH 2?

1. - 42. - 2

3. neutral

4. + 2

5. + 4

Consider the peptide ALRKRG. What is the charge


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p p g

on the peptide a pH 2?

1. - 4

2. - 2

3. neutral

4. + 25. + 4


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Practice Problem


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What is the charge on the following peptideat pH 3, 7, 9 and 11? AHYNERL

Use the pKa values given on slide no. 52.What is the approximate isoelectric point of

this peptide?

Module 2 - Topic 1 - Protein Structure & Function Cont.

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