PROTEINS

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

Time for brain exercise: What is kDa?

Da = ? 1.6611027 kg

Acidic Amino acids Get negatively charged

Glu (G) pKa = 4.3 Above 4.3 pH it will be vely charged

Basic Amino acids Get positively charged

Lys (K)
pKa = 10.5 Below 10.5 pH it will be +vely charged

 Asp

(D)3.9 Glu (E)4.3 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

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

source: Biochemistry by Voet & Voet.

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 (such as a laboratory animal) 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. Heating = denatures protein SDS (sodium dodecyl sulfate) (detergent) = denatures protein Urea (high concentrations denature 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

+
Below pH 6

Above pH 6

Protonated

H+

~6.0 pKa

OH-

Unprotonated

Below pH4.1

Above 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

9.9

2.3

4.3 6

12.4

+vely charged

protonation [H+] pH below pI

pI

deprotonation [OH-]
pH above pI

-vely charge