BIOC

Seven AAs to remember charge

• Asp, Gly, Cys, Tyr

  • Pro: 0

  • Dep: -1

• Arg, His, Lys

  • Pro: +1

  • Dep: 0

3 macromolecules

• Proteins: myoglobin stores O2 in muscle tissue

• Polysaccharides

• Nucleic acids

Polypeptide vs Oligopeptide

P: many amino acids, usually a complete protein

O: few amino acids, usually a fragment

Polarity Defined as

Different electronegativities (tendency to hold bonding electrons)

Hydrogen bond acceptors vs donors

• Acceptors: electronegative atom with available lone pair of electrons

• Donor: hydrogen atom covalently bonded to other electronegative atom

Electronegative elements

O>N>S>C=H (C about H)

Henderson-Hasselbalch equation (to find % dep and pro)

pH = pka + log(Dep/Pro)

Dep = X

Pro = 1 - X

Hydrogen bond donor & acceptors (aa)

Donors: Arg, Lys, Trp

Acceptors: Asp, Glu

Both: Ser, Thr, Asn, Gln, Tyr, His

Column chromatography

buffer on top then separation occurs

Partition Chromatography

• Mobile phase: silica gel (polar)

• Stationary phase: liquid solvent/buffer

• NON polar amino acids move faster

Thin Layer Chromatography

Thin sheet of silica

• Sheet: polar

• Buffer/liquid: non-polar

Elution volume

Volume of buffer needed to move through column

Affinity chromatography

Tagged proteins bind to ligand

Immobilized metal affinity chromatography

His binds to Ni2+ or Co2+

• Resin contains Ni2+

• Imidazole elutes bound protein

• One step purification

Gel filtration (molecular exclusion chromatography)

Gel filled with pores

Large molecules pass through first

Electrophoresis

Movement of charged particles in an electric field

• Polyacrylamide gel: sodium dodecyl sulfate (SDS) surrounds protein with negative charge

• Isoelectric focusing: separate by isoelectric point (when net charge=0). Then increasing pH deprotonates aa and they move to + electrode

• Two dimensional gel: includes both methods

Mass Spectrometry

To identify proteins

Larger particles move more slowly

Specific Activity equation

Specific Activity = Enzyme Activity/Total Protein

Step by step purification of a protein (3)

1. Enzyme activity decreases

2. Total protein decreases

3. Specific activity increases

(specific activity = measurement of purity)

1 Dalton to g/mol

1:1 ratio

1 Da = 1g/mol

Log (molar mass) is the _ axis and elution volume is the _ axis

Log (mm): Y

EV: X

y = mx + b

Protein structure hierarchy

• Primary: linear

• secondary: a-helical or b-sheet

• Tertiary: myoglobin

• Quaternary: Hemoglobin

Protease function

catalyzing hydrolysis (H2O is a poor nucelophile)

What does a nucleophile do

attack/break bonds

Sanger sequencing

N-terminal tagging: tags first aa

but then first aa is lost

Perh Edman sequencing (2 steps)

1. Coupling: Weak base

2. Cyclization: anhydrous acid (except Gly could cyclize early)

Trypsin binds & cuts:

Arg or Lys (unless pro follows: to right)

Cuts right side

Chymotrypsin targets:

Phe, Tyr, Trp

CnBr (Cyanogen Bromide) attacks:

S of Met

Met → Hse (homoserine: serine w extra Ch2)

a-helix and Å/turn

3.6 aa per turn of helix

5.4 Å per turn

So 5.4/3.6 = 1.5 Å (distance between amino acids, or amino acid every 1.5 Å)

Parallel vs antiparallel B-sheet

Parallel: angled bonds:

• Strands run in same direction

Antiparallel: parallel bonds → better alignment → stronger

• Strands run in opposite directions

B-sheet aas and breaker aas

B-sheet: VIT C WYF

Breakers: GPS ND

Collagen: triple helical shape (what aas)

G-P-X

Stabilizing interactions in protein structure (4)

• Ion pairs (+/-)

• H-bonds

• Disulfide bonds (between cys)

• Van der Waals (close interactions)

3 things an atom with a lone pair of electrons can do

1. Accept hydrogen bonds

2. Act as a base (capture H+)

3. Nucleophile: making a bond by sharing e-

In a-helix: h bonding occurs:

at every 5th aa

So atom n H bonds with atom n + 4

Denaturation of protein can be done by:

heat, solvents, detergents (SDS)

a vs b keratin

a: a helix

b: fibroin → antiparallel b sheet

What drives protein folding

hydrophobic effect

• hydrophobic (non-polar) aas stay inside

• hydrophilic (polar) aas like outside

5 tertiary structures

• a-helix bundle: np sides face each other

• B-sheet: antiparallel Bs

• parallel B-sheet: B-a-B-a alternating structure

  • B-strands same direction, np side of sheet faces np side of a-helix

• a-B-barrel (parallel): a-B sheet when helicases on outside

• a-B sandwich (parallel): np B-sheet between 2 a-helix

Disulfide bond

• SH of Cys reacts with O2 (oxidation) → condensation rxn

R-SH & SH-R + O2 → R-S-S-R + H20

Urea & 2-mercaptoethanol

Denatures protein

• Urea: weakens hydrophobic effect: unfolds (denatures) protein

• 2-mercaptoethanol: reducing agent: restores disulfide bond to Cys

After removing both, protein regains native state (renaturation)

Elastase binds ____ and ____ best

Gly, Ala

Arrhenius Equation (rate)

Rate = pze^-(Ea/RT)

• P probability factor (orientation increases)

• Z collision frequency (proximity increases)

• R constant

• T temperature

• Ea activation energy

4 types of catalysis

• Nucleophilic: provides better nucleophile

  • e.g. Cys-S, His-H or Asp/Glu COO- (removing H+)

• Electrophile:

  • Using electron seeking group (electrophile) to remove e- from substrate

• General Base: Removes H+

• General Acid: Donates H+

Prefixes of centi, milli, micro, none (g), kilo

Centi: -2

Milli: -3

Micro: -6

none: 0

kilo: 3

2 Absorbance equations

Io = intensity of light

I = Measured intensity (transmitted light)

E: extinction coefficient (L/mol/cm)

c = sample concentration (mol/L

L = sample thickness (cm)

4 Enzyme catalysis equations

1. Rate of reaction: change in substrate (product) over time

   1. (mol/L min)

2. Enzyme activity: moles of substrate over time

   1. rate x volume (mol/min)

3. Specific activity: enzyme units/mg total protein

   1. Enzyme activity/total protein (mol/min mg)

4. Molar activity: activity per mole of enzyme

   1. Specific activity x enzyme molar mass (1/min)

Rate equations for vo, vmax k1 & k-1

vo (initial rate) = k2(ES)

vmax: k2(E)t

k1 (forward) = k1(E)(S)

k-1 (reverse) = k2(ES) + k-1(ES)

equation at steady rate & km

(k-1 +k2)(ES) = k1(E)(S)

let km = (k-1 + k2)/k1

so km(ES) = (E)(S)

let (E) = (E)t - (ES)

then leads to (ES) = (E)t(S)/(km + (S))

let vo=k2(ES)

then leads to vo=k2(E)t(S)/(km + (S))

let vmax=k2(E)t

then leads to vo= Vmax(S)/ (km + (S))

Higher Vmax means

faster reaction rate, better catalysis

k2 is the true __________ and is the ___________________

constant

Turnover number

km is the concentration of (S) at which rate vo is equal to _____ of the Vmax

50%

If (S) = km, then vo/Vmax = 0.5

Km definition

[S] at ½ Vmax

Double reciprocal plot equation

1/Vo = (Km/Vmax)(1/(S)) + 1/Vmax

y = mx + b

slope: km/Vmax

y-int: 1/Vmax

x-int: -1/km

Competitive vs Non-competitive equilibrium

Competitive: inhibitor can only bind to (E)

• km increased

Non-competitive: inhibitor can bind to (E) or (ES)

• Vmax decreased

5: very polar aas

4 polar aas

5 mod polar aas

6 non polar aas

Very: Asp, Glu, Arg, His, Lys

Polar: Ser, Thr, Asn, Gln

Mod: Gly, Cys, Pro, Trp, Tyr

Non: Ala, Val, Leu, Ile, Phe, Met

Competitive vs Non-competitive reciprocal graphs

K’m equation

Which amino acid’s N-terminal is unavailable to H-bond

Proline

Most mobile in thin layer chromatography

non polar: elute first and move furthest

SDS gel electrophoresis to saparate amino acids by

SIZE

dithiothreitol/performic acid/Urea/2-mercetethanol

dithiothreitol: reducing agent, disulfide bonds → cys

performic acid: oxidizing agent: disulfide bonds → cys → cysteic acid

Urea: weakens hydrophobic effect

2-mercaptethanol: reducing agent, disulfide bonds → cys

Benefit of measuring initial rate vo at t=0

[P]=0 so reverse reaction can be ignored