Biochem Amino Acids And Proteins

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aliphatic amino acids

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Valine
alanine
glycine
proline
isoleucine
leucine

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aromatic amino acids

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phenylalanine
tyrosine
tryptophan

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sulfur containing amino acids

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methionine
cysteine

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alcohol amino acids

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threonine
serine

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acidic amino acids

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aspartate
glutamate

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basic amino acids

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histidine
lysine
arginine

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amide amino acids

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asparagine
glutamine

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F

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Phenylalanine (Phe)

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G

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Glycine- Gly

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A

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Alanine- Ala

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V

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Valine- Val

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L

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Leucine- Leu

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I

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Isoleucine- Ile

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P

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Proline- Pro

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Y

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Tyrosine- Tyr

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W

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Tryptophan- Trp

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M

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Methionine- Met

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C

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Cysteine- Cys

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S

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Serine- Ser

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T

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Treonine- Thr

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D

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Aspartate- Asp

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E

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Glutamate- Glu

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H

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Histidine- His

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K

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Lysine- Lys

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R

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Arginine- Arg

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N

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Asparagine- Asn

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Q

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Glutamine- Gln

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what is a codon?

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a set of three bases along the DNA strand that determines the identity of one amino acid within the protein sequence.

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Ph-OH

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Phenols

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R-SH

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Thiols

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R-NH2

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Amines

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What are the essential amino acids?

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VRH MILK FTW

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Disulfide linkages between individual “cysteines” are called...

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cystines

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Secondary Structure

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alpha – helix & beta - sheet

Both of these secondary protein structures are stabilized by hydrogen bonding between the carbonyl oxygen atoms and the nitrogen atoms of amino acids in the protein chain.

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Anfinson enzyme

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Ribonuclease-
Anfinson used two chemicals to disrupt the enzyme’s 3° structure [DENATURATION ]

urea - disrupts hydrogen bonds
β-mercaptoethanol – reduces disulfide bonds

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Anfinson’s Experiment

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Experiment #1:
Add both urea and β-mercaptoethanol to a solution of enzyme. Activity is lost.
Remove urea by dialysis; then remove β-mercaptoethanol by dialysis. Activity is recovered 100%!
Experiment #2:
Add both urea and β-mercaptoethanol to a solution of enzyme. Activity is lost.
Remove β-mercaptoethanol by dialysis; then remove urea by dialysis. Only ~1% of activity is recovered.

Experiment #3:
Add β-mercaptoethanol to the solution from Exp.#2. Then, remove urea by dialysis;
Finally, remove β-mercaptoethanol by dialysis. Activity is recovered 100%!

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differential centrifugation

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centrifugation at various g-forces
- nuclei are easily separated from mitochrondia, since nuclei spin down at relatively low g-forces. Soluble proteins must be centrifuged at very high forces to spin them out of solution.

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Salting out

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Increasing ionic strength by adding salts such as ammonium sulfate can cause proteins to precipitate. Centrifugation separates the precipitated proteins from the proteins that are still soluble at a given ionic strength.

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Dialysis of proteins

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separated on the basis of their size. Large proteins can be separated from smaller ones via dialysis

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Gel-Filtration of proteins

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separates proteins on the basis of their molecular weights.
-Small pores in the gel allow smaller molecules to become trapped in the matrix, slowing their flow through the column. Larger molecular weight proteins move more rapidly.
-large MW proteins elute from the column before the smaller ones

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Ion Exchange of proteins

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separated on the basis of their net charge.
-Charged proteins bind to ion exchange resins that have opposite charges.
-Changes in pH or salt concentration disrupt this binding, allowing the proteins to elute from the column.

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Affinity Chromatography of proteins

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Some proteins have high affinity for certain chemicals or functional groups.
-If a special column packing material is prepared that contains these high-affinity chemicals, the protein of interest will selectively bind to the column.
-Often, antibodies against the protein of interest will also be immobilized on the gel to bind to their target proteins.
-Affinity chromatography is a very powerful separation technique.

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Electrophoresis of proteins

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Proteins contain numerous ionic charges (+/-), depending on pH. When placed in an electrical field, the proteins move at different rates
-larger MW proteins move slower that smaller proteins, due to steric hindrance.
-To add negative charge to the surface of proteins, “SDS” [sodium dodecylsulfate] detergent is added

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what does SDS-PAGE stand for

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sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Ultra-Centifugation of proteins

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By spinning proteins at ultra-high g-forces in an “ultra-centrifuge,” properties such as their partial specific (molecular) volume, their shape, density may be studied.
-The smaller the “Svedberg”value (“S”), the slower a molecule moves in a centrifugal field

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Zonal-Centifugation of proteins

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The use of “solution gradients” help by allowing the protein to move to its “sedimentation equilibrium.

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MALDI TOF

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Matrix-asisted Laser Desorption-ionization Time of Flight
-Mass Specrometry
-very precise determination of the molecular weights of proteins.

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power based on the ability to punish is what form of power?

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coercive

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Fluorescamine

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Detects as little as one nanogram of amino acids

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Amino Acid Composition of proteins

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The protein is totally hydrolyzed (e.g., 6N HCl at 110°C for 24 hours). Quantitation of each amino acid is determined by ion-exchange chromatography. Normally, the hydrolyzed amino acids are visualized by reacting with Ninhydrin, turning them an intense blue color (except proline, which turns yellow).

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Automated Edman Degradation of proteins

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phenyl isothiocyanate is used which sequentially reacts with amino-terminal residues, releasing “PTH-amino acids
-is limited to less than 50 residues, if longer polypeptides are encountered, the protein must be broken down into shorter segments.

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CNBr

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cleaves peptide bonds on the carbonyl side of methionine residues

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trypsin

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catalyzes the hydrolysis of peptide bonds on the crboxyl side of either lysine or arginine

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Carboxypeptidase A

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cleaves amino side of C-termical amino acid (not R, K or P)

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X-Ray Crystallography

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reveals 3-D structure in great detail by measuring distances between atoms in a protein crystal
-yields electron-density maps that can be used to determine the spatial arrangement of the atoms in the molecule

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NOESY

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Nuclear Overhauser Enhancement Spectroscopy
-NMR can measure interactions between atoms that are relatively close to one another in the tertiary structure
-However the atoms must be close to each other, as this energy transfer decreases with the radius to the SIXTH power!

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Indirect ELISA

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Detects the presence of an antibody
-color formation is proportional to the amount of specific antibody

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Sandwich ELISA:

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Detection and Quantitation of Antigen
- color formation is proportional to the amount of antigen

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Western Blots

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Detect Proteins in Gels
-Separated proteins in an SDS PAGE gel are transferred to a polymer sheet. Then antibodies identify the location of the desired protein

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Purines

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Adenine
Guanine

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Pyrimidines

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Cytosine
Uracil
Thymine

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Watson and Crick publication highlights

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-two helical chains each coiled round the same axis
-phosphate diester groups joining ß-D-deoxyribofuranose residues with 3',5' linkages
-two chains run in opposite directions

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Melting

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Melting” DNA separates the two helical chains by disrupting the hydrogen bonds between bases.
At the “melting temperature” (Tm), the bases separate and “unstack.” This results in increased absorption of UV light:

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DNA in chromosomes is tightly bound to proteins called

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histones

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Histone octamers surrounded by about 200 base pairs of DNA form units called

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nucleosomes

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Chromatin

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tightly-packaged, highly-ordered structure of repeating nucleosomes