presentation1 biochem

8/13/2019 Presentation1 Biochem

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1) Draw the general structure of an amino acid at pH 7.0.


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1) Draw the general structure of an amino acid at pH 7.0.

H+

R CH

CO O

NH3


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- Form depends on the pH:

pH 6-7 pH < 2pH > 9

they are amphoteric (react with acid and base)

H+R CH

COOH

NH3

R CH

CO O

NH3

OH

R CH

CO O

NH2


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2) Draw the structure of glutamic acid at pH 2, 7, 11


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2) Draw the structure of glutamic acid at pH 2, 7, 11

In an acid solution there are many protons H + so: COO- gains H+ and is

deionized (no charge)


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pH 7

COOH is ionized ie. Loses

H+ so now charged

pH 11

In a basic solution there are many

OH- (hydroxide ions), H3N is

deionized ie. loses H+ so now no

charge


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3) Give the name & one letter symbol for each of the following;

(a) an amino acid which has H as its R group.

(b) an aromatic amino acid

(c) a hydrophobic amino acid

(d) a polar amino acid

(e) a cyclical amino acid

(f) a positively charged amino acid

(g) an amino acid that can covalently bond to another identical amino acid.


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3) Give the name & one letter symbol for each of the following;

(a) an amino acid which has H as its R group.

Glycine (Gly) G

(b) an aromatic amino acid

Phenylalanine (Phe) F Tyrosine (Tyr) Y Tryptophan (Trp) W


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(c) a hydrophobic amino acid

Valine (Val) V Alanine (Ala) A Isoleucine (Ile) I Leucine (Leu) L

Are non-polar and dont interact with water (which is polar). Are aliphaticie. Have open chain hydrocarbons


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(d) a polar amino acid Glycine (Gly) G

Serine (Ser) S

Methionine (Met) MThreonine (Thr) T

Glutamine (Gln) Q

Cysteine (Cys) C

Asparagine (Asn) N


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(e) a cyclical amino acid

Proline (Pro) P

(f) a positively charged amino acid

These will be the basic amino acids

Histidine (His) H Lysine (Lys) L Arginine (Arg) R


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(g) an amino acid that can covalently bond to another identical amino acid.

Cysteine Cysteine (Cys) C with a disulphide bond


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4. What useful property of aromatic amino acids is utilised when studying

proteins in solution ?


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4. What useful property of aromatic amino acids is utilised when studying

proteins in solution ?

Aromatic amino acids can absorb UV light at 280nm. This is also how we

can detect and quantitate aromatic amino acids.

Absorption Spectrum for Cytochrome "C"

0

0.5

1

1.5

2

2.5

3

360 385 410 435 460 485 510 535 560 585 610 635 660

Wavelength (nm)

Absorbance

Cytochrome :"C" After sodium ascorbate added Baseline


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Sample requirements:sample to be analysed must contain onlyone absorbing component for which the calibration has beenperformed. If sample is a solution, a pure sample ofthe solvent should be used as a blank.

concentration

A

bsorbance

Linear relationship

between absorbance, A,

and concentration

A = -log(T)

A = ecl


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5. What two pieces of experimental evidence prove that polypeptide

conformation is dependent on primary structure?


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1. Proteins will reform back into native confirmation if secondary,

tertiary and quartenary structure unfolded (denatured) but not if

primary sequence disrupted.

Denaturation- the unraveling of a proteins structure. Break down all

secondary, tertiary and quaternary structure. The disorganized protein willno longer act as intended.

When this occurs, protein strands will clump together - coagulate.

Examples - frying an egg

- Low pH in stomach

Temperature or pH outside the normal range can both cause denaturation.


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Denaturing a protein

heat

or

acid

heat

or

acid

denatured coagulated


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Renaturation

Providing the

1ostructure

remains in

place, then

protein will

often refold


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2. Site specific mutagenesis: mutation in just one residue can change

structure and function of protein. Widely used to determine role of

individual residues in proteins. Conservative change eg. Hydrophobic

aa replaced with other hydrophobic aa usually results in similar

structure.

Eg. Sickle cell disease results due to Valine for Glutamine at residue 6 in

the Hbb chain.


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6. Discuss the 3-D conformation of a protein. In particular how is this related to activity.


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Sequence of amino acid residues in

polypeptide, e.g.

-Asp-Leu-Met-Thr-Ser-Tyr-

or

DLMTSY

-Hydrolysed to amino acids

-Polypeptide sequencing

e.g. Edman degradation

N-terminus

C-terminus

Primary Structure

6. Discuss the 3-D conformation of a protein. In particular how is this related to activity.


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Secondary Structure

Long chains of amino acids will commonlyfold into a regular repeating structure,

such as - helix or - pleated sheet

Structureis a result of:

- peptide bond angles

- R group interactions

- hydrogen bonding between amino acidswithin the protein.


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RGroup Interactions

R-Groups interact in a variety of ways:

- Hydrogen bonding- Ionic linkages

- Metal ion coordination

- Hydrophobic interactions- Covalent bonds

- Steric Hindrance


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-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

|

NEvery amide hydrogen and

carbonyl oxygen is involved

in a hydrogen bond.

R-Groups are on the outside

of the helix

Properties of an -helixinclude strength and low

solubility in water.


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-Pleated sheets

Another secondary structure for protein.

Hydrogen bonding between adjacent sheets of protein.

C|

R

R|

C

R|

C

R|

C

R|

C

C|

RC|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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Turns and Loops

Turn- 4 residues.

- Reverse direction of the main polypeptide

chain (several subtypes).

- Connect regions of more regular secondary

structure (-helix, -sheet) Loop- 6-16 residues. (Random Coil)

- Continuous segment of a polypeptide chain.

Both have no extended secondary structure. They

serve to tie together other units.


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Protein Tertiary Structure

Interactions between amino acid residuesand environment results in a proteintaking on a stable, compact arrangement.

Central dogma of protein folding

The primary structure determines thetertiary structure

Proteins with a unique primarystructure tend to fold spontaneously into adistinct tertiary structure.


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Protein Tertiary Structure

Folding occurs asa stepwiseprocess.

Only the final formis biologicallyactive.

This final form isthe proteinsnativeconformation


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Quaternary structure

of proteins

Many proteins are not single peptide strands.

They are: - combinations of several proteins

- aggregate of smaller globular

proteins.

Conjugated protein - incorporate another type of group that performs a

specific function.

prosthetic group eg; Haem


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Multi Subunit Proteins

HomomultimersIdentical Subunits

eg Homodimer

Heteromultimers- Different Subunitseg Heterodimer

Subunit Association is usually non-covalent

Association and Stabilisation due to samefactors that hold tertiary structure


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Hydrophobic attractions

- Attractions between R groups of non-polar amino acids.

Hydrogen bonding

- Interaction between polar amino acid

- R groups.

Ionic bonding- Bonding between + and - charged

amino acid R groups.

Disulfide LinkagesCys S=S Cys

Subunit Stabilisation


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Summary of protein structure

primary secondary

tertiary quaternary

H O

| ||

H2N - C - C

|R

H

|

N - C - COOH

| |H R

H O

| ||

- NH - C - C -

|R


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7) Compare and contrast the structure and function of myoglobin and haemoglobin


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7) Compare and contrast the structure and function of myoglobin and haemoglobin

Both oxygen binding proteins, Myoglobin for O2storage, Haemoglobin for O2

transport. Myoglobin is a single polypeptide chain, haemoglobin is a tetramer of 2

different subunits ( & ). Both similar tertiary structure but only haemoglobinhas quarternary structure.

O2 binding to myoglobin is hyperbolic

O2 binding to hemoglobin is co-operative allosteric

When plenty of O2 (lungs),stronger binding capacity. Binding of an O2 results in

conformational change and promotes binding of next. When low O2

as in tissues, weak

binding capacity.


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8) What special features characterize transmembrane proteins?


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8) What special features characterize transmembrane proteins?

Transmembrane proteins, such as cell surface receptors or ion channels, have exposed

hydrophobic residues that allow them to embed in the hydrophobic core of lipid bilayer

membranes, such as the plasma membrane. They are often characterized by membrane

spanning alpha-helices, i.e. TM7 receptors.

Membrane receptors

transduce external

signal from large

signalling moleculesto cell interior,

without signalling

molecules entering

cells

e.g. 7

transmembrane helix

(7TM) receptors, such

as -adrenergic

receptor

presentation1 biochem

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