Biochemistry Exam 2

Pka of Tyr

10

Pka of Cys

8

Pka of Asp

4

Pka of Glu

4

pKa of Arg

12

pKa of His

6

pKa of Lys

11

Phenyl groups pka

10

Counting carbons

Carbonic acid is 1

isoelectric point (pI) of a protein pH

the pH at which the net charge of the protein is neutral

PI of basic amino acids

8-9

PI of acidic amino acids

4-5

Charge from amino acid composition pH

Matters with the amine and carbonic acid

What is the charge of histidine at pH less than 6?

Positive charge

What is the charge of histidine at pH greater than 6?

Neutral charge

Protein columns

a tube filled with a (resin) used to separate and purify proteins from a mixture

Fraction collector

Spot to hold test tubes so the outlet can collect the drops

UV detector

Trp is in UV to detect the proteins

ion exchange chromatography

molecules separated based on net surface charge

What does ion exchange chromatography depend on

pH and salt concentration

cation exchange chromatography

Positive proteins stick to negative beads, only negative proteins go through

anion exchange chromatography

Negative proteins stick to positive beads, only positive proteins go through

affinity chromatography

Purify and isolate a specific type of protein from within a mixture of proteins based on their affinity to bind to specific molecules

What is a tag in genetic engineering?

A sequence that can be genetically fused to a protein.

What is the purpose of a tag in protein purification?

To allow one to purify expressed fusion protein by nickel affinity chromatography.

Size exclusion chromatography (gel filtration)

relies on porous beads; larger molecules elute first because they are not trapped in small pores

Polyacrylamide gels (PAGE)

for very small DNA fragments and ssDNA; polymerization requires catalyst

Gels are crosslinked

Pockets in gel allows for separation

more crosslinks

Smaller pore size

More % in gel

more variability for different protein sizes

Picking gel for PAGE

Protein near the middle

4-15%

4% top and 15% bottom

15%

Stopping proteins from running off the bottom

Sample buffer

Buffer used to prepare samples for electrophoresis.

Sample buffer glycerol

Sit in the lanes, Makes proteins heavy enough 

Analysis of SDS-PAGE

Pure = single band

SDS

Coats proteins with a negative charge

WHy heat a SDS-PAGE gel

Heating breaks hydrogen bonds, covalent stay

Consensus sequence

Align protein sequences

X on Consensus sequence

any animo acid

Blue on Consensus sequence

positive charge

Red on Consensus sequence

Negative charge

Size on Consensus sequence

larger = more common

{} on Consensus sequence

could be anything but this, Usually grouped together

Homologs

A shared origin

Orthologs

homologous genes separated by a speciation event

Paralogs

homologous genes within a single species

oligopeptides

chains of fewer than 10 or 15 amino acids

Primary structure

amino acid residues are connected via covalent bonds (peptide bonds) and disulfide bridges (Cys-Cys)

rise/amino acid of a-helix

1.5

Width of a-helix

5A

A-helix h-bonds

every i, i+4

A-helix charges

Opposite change on the same side = more stable

Good amino acids to form a-helix

Ala, Arg, leu, Lys, Met, Ile

# for formation of a-helix

Low

C-term in a-helix

negative, has positive side chains

N-term in a-helix

positive, has negative side chains

Fraction of residues of a-helix

1/4

N & C direction on beta sheet

N to C

Antiparallel B-sheet

H-bonds are straight

Φ & Ψ of B-sheet antiparallel

Φ = -139

Ψ = + 135

Parallel B sheet

H-bonds are diagonal

Length per aa B-sheet

3.5 A

B-turn

Where backbone changes direction 180 in 4 a.a

Hydrogen bonds Main Participants

Polar side chains, backbone

Hydrogen bonds Typical Location

Surface & interior

Hydrophobic interactions Main Participants

Nonpolar side chains

Hydrophobic interactions Typical Location

Inside protein

van der Waals (dispersion) Main Participants

All atoms (nonpolar)

van der Waals (dispersion) Typical Location

Packed core

Ionic interactions (salt bridges) Main Participants

Charged side chains

Ionic interactions (salt bridges) location

Surface

Keratin Amino acids

Ala, Val, Leu, Ile, Met Phe ( all hydrophobic)

Keratin Structure

α-helices that twist together to form coiled coils, Right handed

Hydrophobic residues in Keratin

that align inward for stability

More Disulfide bonds in keratin

stronger

Function of keratin

Strong, flexible fibers: the α-helix and coiled-coil design gives tensile strength and elasticity

Collagen

structural protein found in the skin and connective tissue

Collagen helix

left handed

Collagen aminos acids

Gly-X-Y

X= proline

Y = 4 hydrophobic amino acids

Collagen function

High tensile strength the rope-like triple helix resists stretching

Crosslinks in collagen

gives long-term stability and durability

Silk structure

antiparallel beta sheets

Side chains of silk

Gly and ala

Does silk stretch

Doesn't stretch (it is already elongated)

Φ is near

Nitrogen

Ψ is near

Carbonic acid

Right-handed α-helix Φ & Ψ

φ ≈ −60°, ψ ≈ −40° (cluster near (−60, −40))

β-sheet α-helix Φ & Ψ

φ ≈ −135°, ψ ≈ +135°

Protein

Macromole with 1+ polypeptide chain

Enzyme

protein that acts as a biological catalyst

Substrate

A specific reactant acted upon by an enzyme

Ligand

Molecule that binds to a receptor

where is Myoglobin found

muscle cells

Myoglobin's function

stores and holds oxygen

myoglobin structure

8 a-helixes, Compact allows efficient O₂ storage

What does myoglobin do with Fe?

Preventing oxidation of Fe 2+ to Fe 3+

Heme group of myoglobin

porphyrin ring + iron

Ligand of myoglobin

Oxygen gas

Hydrophobic pocket of Myoglobin

keep Fe 2+, bind O2