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Proteins are made of ?

Amino Acids
Total amino acids = 20

20 standard amino acids

20 standard amino acids


At physiological pH amino acids are charged as shown on left
Can act either as acid or base
Soluble in water

Why?

Protein are polypeptide

Peptide bond
Partial double bond character

The amide structure has two resonance contributors:

Therefore, rotation around this bond is restricted.


Thus, the peptide unit is a planar, rigid structure and
rotation in the peptide backbone is restricted to the
bonds involving the a-carbon

Peptide Bond

Time for brain exercise:


What is kDa?
Da = ?1.661027 kg

Acidic Amino acids


Get negatively charged

Glu (E)

pKa = 4.3
Above 4.3 pH it will be vely charged

Basic Amino acids


Get positively charged
Lys (K)

Lys (K)

pKa = 10.5
Below 10.5 pH it will be +vely charged

pKa values
Acidic amino acids
Asp (D)3.9
Glu (E)4.3

Basic amino acids


Arg (R)12.0
Lys (K)10.5
His (H) 6.08 (+vely charged below 6.08)

Protein and Amino acid pKa values


pKa values of amino acid side chains play an important
role in defining the pH-dependent characteristics of a
protein.

The pH-dependence of the activity displayed by enzymes


and the pH-dependence of protein stability, for example,
are properties that are determined by the pKa values of
amino acid side chains.

Hydrophobic/Nonpolar

Polar

Hydrophobic/Nonpolar

Absorption Spectroscopy of proteins


Used to measure protein concentration

Wavelength used 280 nm


Proteins in solution absorb ultraviolet light with absorbance
maxima at 280 and 200 nm.
Why?
Amino acids with aromatic rings (Tyrosine and Tryptophan) are
the primary reason for the absorbance peak at 280 nm.
Peptide bonds are primarily responsible for the peak at 200 nm.

UV Spectrophotometer uses Quartz (crystalline silica) cuvette

Beer Lambert Law


A cl
A= absorbance
c= concentration
l = path length
A = cl.
e = molar absorbtivity coefficient (Molar Extinction Coefficient ) with
units of L mol-1 cm-1

Means how strongly a substance absorbs light

Intrinsic property of substance, depends on chemical nature.


A = no units
c = mol L-1
l = cm

Biocatalyst
Biochemical reactions
Industrial application: Proteases/Lipases = added detergents
Cellulase = stone wash jeans

Pharmaceutical Industry

pH
pKa
Isoelectric point (pI)
Charge on a polypeptide

Why is protein folding/native conformation of


protein important?

Genetic blood disorder characterized by red blood cells that assume an


abnormal, rigid, sickle shape.
Mutation in hemoglobin

Sickle-cell hemoglobin: a surface polar-to-hydrophobic


mutation that lowers solubility
Glu to Val mutation at 6th amino acid
causes self-association and
polymerization
mutation leads to
hydrophobic
interaction
between
hemoglobin
tetramers

Phe 85
Val 6

Leu 88
fibril
formation
at high
concentration

source: Biochemistry by Voet & Voet.

picture of
sickle-cell hemoglobin
fibrils spilling out of
a distorted, ruptured
erythrocyte

Misfolded
protein due to
mutation

Human CJD disease: Cruetzfeld-Jakob disease


Cattle: Mad cow disease
Prion diseases are transmissible from host to host of a single species and,
sometimes, even from one species to another
Destroy brain tissue giving it a spongy appearance

If protein is not in its native 3D conformation


That means it is denatured

Protein/enzyme will lose its activity


Remember:
When purifying a protein one has to be careful that it does not
get denatured.
What chemical reagent you use for purification is important.
What pH of buffer you use important.

= Heating = denatures protein


= SDS (sodium dodecyl sulfate) (detergent) = denatures protein
= Urea (high concentrations denature proteins) = breaks Hbonds + hydrophobic interactions

How to purify and identify proteins?


Separation techniques make use of protein properties
Properties of protein depend on the amino acid sequence

(pI)

The net charge (the algebraic sum of all the charged


groups present) of any amino acid, peptide or protein,
will depend upon the pH of the surrounding aqueous
environment.

As the pH of a solution of an amino acid or protein


changes so too does the net charge.
When the net charge of an amino acid or protein is zero
the pH will be equivalent to the isoelectric point: pI.

Histidine

Above pH 6

Below pH 6

Protonated

H+

~6.0
pKa

OH-

Unprotonated

Above pH4.1

Below pH4.1

Protonated

H+

pKa

OH-

Unprotonated

+
Protonated

H+
H+

12.5
pKa

OHOH-

0
Unprotonated

The peptide bond is not charged, however


you still have a + at the N-terminal, - at the Cterminal, and any charges on side chains

An amino acid can not exist as COOH/NH2


because any pH low enough to protonate the
COO- group would also protonate the NH2
Or pH high enough to deprotonate the NH2
group would also deprotonate the COO-

9.9

2.3

4.3
6

12.4

+vely charged

protonation
[H+]
pH
below
pI

pI

deprotonation
[OH-]

-vely charge

pH
above
pI