Exam I

What is Ka and how does it relate to pKa?

Ka: describes acid strength

pKa: -log(Ka)

What determines whether a proton comes off?

How stable is Conjugate Base (A-)

What is pH

-log[H+]

Henderson Hassel-Bach

what is the main idea behind it

pH = pKa + log [A-]/[HA]

when pH = pKa

then [A-] = [HA]

deprotonated = protonated

When are buffers most effective

at their pka range (pH=pKa)

buffering range = ± 1

How do you determine the CHARGE of the ionizable group based on pH & pKa

pH < pKa → protonated form dominates

pH > pKa → deprotonated form dominates

then determine the charge of that form!

Titration Curve

• equivalence pt

• ½ equivalence pt

equivalence point = 1eq base has been added for each ionizable proton→ fully deprotonated

½ equivalence point: the pt where [protonated] = [deprotonated] (pH = pKa)

What is a Nucleotide

vs a Nucleoside

Base + Sugar (Ribose or Deoxy) + Phosphate(s)

vs

Base + Sugar

Ribose vs Deoxyribose

• why does this matter

Ribose has C2’ -OH, Deoxy does not.

This -OH makes RNA much less stable because it means that it undergoes alkaline hydrolysis of phosphodiester bonds.

this means that in basic conditions the -OH can nucleophilic attack and break the backbone.

RNA → much faster hydrolysis

DNA → much more long-term stability

How to Number Nucleotide Sugars?

PRIME DESIGNATION

C1’ → forms glycosidic bond w nucleobase

C2’ → tells you DNA v RNA

C3’ → OH forms next phosphodiester bond

C’5 → attached to phosphate

What is a Sugar Pucker?

What are the conformations?

• non-planar sugar conformation (due to steric strain)

e.g.

ENDO (atom out of plane is on same side as C5’)

or

EXO

4 standard conformations:

• C2’ endo/exo

• C3’ endo/exo

FREE nucleotides are more free to switch between conformations (isoenergetic)

Conformation of DNA sugar

C2’ endo sugar puckers

Why are the DNA base pairs complementary

• Their shapes and H-bonding patterns allow specific pairing: A–T and G–C.

How many bonds hold A-T or C-G

A–T = 2

G–C = 3.

What is base stacking

Why analogous to the hydrophobic effect?

Adjacent aromatic bases stack on one another inside the helix, stabilizing DNA.

• Nonpolar base surfaces cluster away from water, while water is excluded from the stacked interior.

H = change in enthalpy (heat content)

S = change in entropy (disorder)

T = abs temp (K)

@ Standard Conditions (1atm, 1M, 298K)

ΔG° = standard Gibbs free-energy change

• R = gas constant = 8.314 J/(mol·K)

• T = temperature in Kelvin

• K = equilibrium constant

@ Standard Conditions (1atm, 1M, 298K)

• at equilibrium?

• ΔG° → standard conditions

• ΔG → actual/current conditions

• Q → what the reaction mixture currently has

How can reactions be driven forward?

Alkaline Hydrolysis

lkaline hydrolysis is the base-catalyzed cleavage of RNA's phosphodiester backbone, where OH⁻ promotes attack by the RNA 2′-OH on the adjacent phosphate.

What are Phosphoanhydride Bonds?

High energy bonds between phosphates

their hydrolysis can provide the energy that drives otherwise unfavorable biochemical reactions.

Beer Lamberts

Abs = E l c

How does absorbance change with concentration?

They are directly proportional.

If you have more molecules, they absorb more light, so absorbance increases.

What is the wavelength for Nucleic Acids?

• why

260nm

• the nitrogenous base

What wavelength do amino acids/ proteins absorb at?

• why?

280nm

• aromatic aas (WYF)

Hyperchromic effect

means more ABSORBANCE at 260nm when double stranded DNA becomes single stranded.

due to less base stacking and h-bond interactions

What is the melting temperature

The temperature where about 50% of the DNA is denatured is the melting temperature (Tₘ).

How to increase melting temperature of DNA

• increase the G-C content (stronger stack/h-bond interactions)

• increase DNA length

What is a zwitterion?

a molecule that has both a positive charge and a negative charge at the same time, but its overall/net charge is zero.

An amino acid has two groups that can gain/lose H⁺:

• Amino group: –NH₂

• Carboxyl group: –COOH

What happens to an Amino Acid at Low pH

lots of H+ floating around, AA becomes protonated (+ charge)

What is pI

The Isoelectric Point (The pH at which an amino acid has a net electrical charge of zero.)

the pI is the average of the two pKa values surrounding the neutral form.

pH of C vs N

• pKa₁ (COOH) ≈ 2.3

• pKa₂ (NH₃⁺) ≈ 9.6

What is the pI of a peptide with NO ionizable side chains?

6

Which amino acid is achiral

What

Glycine

What form of aa do people use

L form amino acids

What does it mean if a peptide has a pI > 7?

BASIC peptide, carries some + charge at pH 7

What does it mean if a peptide has pI < 7?

ACIDIC peptide, carries some - charge at pH 7

What is a Disulfide bond

• 2 Cys OXIDIZE

• Strong COVALENT bond

• stabilizes protein structure

weak 280 abs

Why do amino acids have two buffering regions

As you add base COOH loses H⁺:

Its pKa is around 2.3.

As you add more base, the NH₃⁺ loses H⁺:

Its pKa is around 9.6.

Peptide Bonds

• formed via dehydration/ condensation

• pecifically, it forms between:

  • the carboxyl group (–COOH) of one amino acid

  • the amino group (–NH₂) of another amino acid

What are the ionizable groups and their pKas

RKYCHED!

R - 12

K - 11

Y - 10

C - 8

H - 6

E - 5

D - 4

What does LABILE mean for a peptide bond/

easy to cleave under certain conditions

What is a Protein Sequence Alignment?

Lines up homologous sequences to compare corresponding amino acid positions.

Invariant/ Identical Residue

exact same amino acid.

Similar Residue

Different amino acid with similar chemical properties.

How to calculate % identity?

exact matches / total x 100

How to calculate % similarity?

exact + similar matches / total x 100