Transcript Document

BIOL710: Molecular Biology
Mondays and Wednesdays, 5:30PM
LECTURERS
SUGGESTED TEXT
Mitchell Goldfarb
HN834 772-5289
[email protected]
Paul Feinstein
HN904 650-3169
[email protected]
Biochemistry, 6th edition
Berg, Tymockzo, & Stryer
ISBN: 0716787245
OR
Biochemistry, 4th edition
Stryer
ISBN: 0716720094
Available online from
www.barnesandnoble.com
www.amazon.com
COURSE WEBSITE
http://biology.hunter.cuny.edu/molecularbio/
Scope of the Course
How are biological macromolecules synthesized and assembled?
How do different macromolecules generate the structure of cells?
How do proteins fold to acquire functionality?
How do enzymes catalyze reactions?
How is energy harvested and stored in the cell?
How do pumps and channels store energy and control the chemical
composition of cellular compartments?
Intracellular biochemical signaling by proteins and lipids.
DNA structure and replication.
Transcription and post-transcriptional RNA modification.
Regulation of transcription.
The genetic code and protein translation.
Similarities and differences in processing of genetic information
in prokaryotes vs. eukaryotes
LECTURE 1: pH, pK, Amino Acids, Peptide Properties, pI
Reading: Berg Chapters 1,2
Before being able to discuss the properties of biological
macromolecules, we must first review:
1) Covalent and noncovalent bonding
2) Oxidation and reduction
3) Acid/base chemistry
Elemental Properties
H
Hydrogen
Carbon
Oxygen
H
C
O
Electropositive
Forms 1 covalent bond
Forms 4 covalent bonds
H
H
O
H
C
C
H
H
H
H
O
C
C
Sulphur
N
Strongly electronegative
Forms 2 covalent bonds
S
Weakly electronegative
Forms 3 covalent bonds
Weakly electronegative
Forms 2 covalent bonds
P
In oxidized form
Forms 5 covalent bonds
N
H
H
H
H
R -- O -- P -- O -- H
-
H
O
C
C
phosphate side group
O
H
glycine
H
O
O
H
acetic acid
N
Phosphorus
O
H
H
Nitrogen
ethanol
H
O
C
C
C
H
S
H
O
H
cysteine
Oxidation and Reduction of Organic Compounds and Oxygen
OXIDATION -- Removal of an electron pair from a molecule
REDUCTION -- Addition of an electron pair from a molecule
In any oxidation/reduction reaction, one component (electron donor) gets oxidized,
another component (electron acceptor) gets reduced
H
H
O
H
C
C
H
H
H
-
2e + 2H
+
H
O
C
C
H
ETHANOL (reduced)
H
ACETALDEHYDE (oxidized)
O
O
H
O
O2
H
+
ACCEPTOR
2NADH
DONOR
+ 2H+
H
2H2O
REDUCED
+
2NAD+
OXIDIZED
Weak Acids and Bases
When hydrogen is covalently bonded to an electronegative atom
(typically O or N) in a compound, the hydrogen proton may dissociate.
In this situation:
The protonated compound is called an ACID,
and the deprotonated compound is called a BASE.
Conjugate ACID
Conjugate BASE
H
H
H
O
C
C
H
acetic acid
O
H
H
H
O
H
H
C
C
N
H
H
H
+
hydroxyethylammonium
H
O
C
C
H
H
H
+
H
O
-
acetate
+
H
O
H
H
C
C
H
H
H
N
2-amino-ethanol
H
pH
Water is in equilibrium with its minor dissociation products, H+ and OHWhile concentration of H2O is 55 M, [H+][OH-] = 10-14 M
In a neutral solution, [H+] = [OH-] = 10-7 M
In an acidic solution, [H+] >> [OH-] . E.g. [H+] = 10-3 M , and [OH-] = 10-11 M)
In a basic solution, [H+] << [OH-] . E.g. [H+] = 10-10 M , and [OH-] = 10-4 M)
pH = - log10 [H+]
At neutrality, pH = - log10 [10-7] = 7
Acidic solutions have pH < 7; Basic solutions have pH > 7
Generally speaking
“weakly acidic” means pH = 4 to 5.5
“strongly acidic” means pH < 3
“weakly basic” means pH = 8.5 to 10
“strongly basic” means pH > 11
Strong Acids and Bases
A strong ACID is an INORGANIC compound that FULLY DISSOCIATES into
H+ and its negative counterion in water.
H+
HCl
Cl-
H+
HNO3
HYDROCHLORIC ACID
NO3-
NITRIC ACID
A strong BASE is an METAL HYDROXIDE compound that FULLY DISSOCIATES into
OH- and its positive counterion in water.
NaOH
OH-
Na+
2OH-
Ca(OH)2
SODIUM HYDROXIDE
CALCIUM HYDROXIDE
pH of strong acid and base solutions
pH = - log10 [ACID]
Ca+2
pH = 14 + log10 [BASE]
e.g.
e.g.
pH of 10 mM HCl = 2
pH of 0.1 M NaOH = 13
TITRATION OF A STRONG ACID SOLUTION WITH STRONG BASE
Different amounts of NaOH added to a 10mM HCl solution
pH stays strongly acidic until nearly 10mM NaOH added,
then swings steeply past neutral to strongly basic when NaOH exceeds 10mM
NO CONTROL OF pH IN THE MILD ACID TO MILD BASE RANGE
pH
NEUTRAL
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
0
1
Molar Equivalents of NaOH
2
pKa of an acid/base conjugate pair
H+ +
HA
Acid
Ka =
[H+ ][A- ]
Proton
TAKE LOGARHYTHM
MULTIPLY BY -1
[HA]
ABase
[A-]
pH = pKa + log10
[HA]
IMPLICATIONS FOR ANY PARTICULAR ACID/BASE PAIR
For a group with an acidic pKa, the base conjugate predominates at neutral pH
For a group with an basic pKa, the acid conjugate predominates at neutral pH
When acid and base are at equal concentrations, pH
= pKa
An acid/base pair acts as a buffer of strong acids or bases
in pH range of
pKa + 1.
Titration of a Weak Acid Solution with a Strong Base
The weak acid BUFFERs the effect of the strong base,
keeping pH in the range of the pKa over wide range of base concentratiion
As NaOH is added to a weak acid solution HA, the NaOH converts HA to A
HA + NaOH
-
H2O + Na+ + A-
Since pH = pKa + log [ A- ]/[ HA ] , the weak acid buffers in the range of pKa
7
For acetic acid
pKa = 4.0
100
6
5
2
25
1
0
0
0.5
1
Molar Equivalents of NaOH
pH
-
For unbuffered
0.1 M acetic acid
pH = 2.5
50
3
[A ] (mM)
4
[HA] (mM)
pKa
75
Three Types of Non-Covalent Bonding Influence
Intramolecular and Intermolecular Interactions
ELECTROSTATIC BONDING
Oppositely charged groups are attractive.
HYDROGEN BONDING
A hydrogen covalently bonded to O or N can noncovalently interact with a O or N,
if all three atoms are aligned and at appropriate distance. This is a hydrogen bond.
H
O
R
O
H
O
R
H
H
Three Types of Non-Covalent Bonding Influence
Intramolecular and Intermolecular Interactions
VAN DER WAAL’S FORCES AND HYDROPHOBIC INTERACTIONS
A weak interaction between nonpolar molecular surfaces.
Van der Waal’s forces contribute to favorability of hydrophobic interactions.
The other crucial contributing factor is that interaction between two hydrophobic
surfaces in a solution reduces the hydrophobic surface area and therby
INCREASES the number of water-to-water solvent hydrogen bonds!!!
AMINO ACIDS
pK of carboxylic acid group is ~ 3.0
pK of amino group is ~ 9.5
POLYPEPTIDES
FREE
ROTATING
All polypeptides synthesized
from L-amino acids
FREE
ROTATING
CLASSES OF AMINO ACIDS
All proteins are synthesized from a pool of 20 amino acids
(some additional amino acids are generated by modifications within synthesized polypeptides)
Amino acids can be functionally grouped by properties of side chains (R)
(a few amino acids fit overlap into more than one group)
GROUPINGS
ALIPHATIC -- Side chains participate in hydrophobic interactions
AROMATIC -- Hydrophobic interactions and hydrogen bonding
ACIDIC --
Ionic and hydrogen bonding
BASIC --
Ionic and hydrogen bonding
HYDROXYL -- Hydrogen bonding and sites of phosphate or sugar modifications
AMIDO --
Hydrogen bonding and sites of sugar modifications
SULPHUR -- Hydrogen bonding and sites of oxidative crosslinking
“OTHER” -- Side chains confer specialized turning properties of polypeptide
ALIPHATIC AMINO ACIDS
NOTE:
AROMATIC AMINO ACIDS
Methionine is
also a
sulphurbearing
amino acid
NOTE:
Tyrosine is
also a
hydroxylated
amino
acid
HYDROXYLATED AMINO ACIDS
Ser-, Thr-, or Tyr-OH in protein can undergo phosphate addition
Ser- or Thr-OH in protein can can be site for carbohydrate addition
(termed O-linked glycosylation)
ACIDIC AMINO ACIDS
Carboxylic acid side chain in Asp and Glu
has pKa ~ 4.5 and carries
a full -1 charge at neutral pH
AMIDO AMINO ACIDS
Asn is amidated version of Asp
Gln is amidated version of Gln
Asn and Gln are NOT charged,
but are higly polar
NH2 group on Gln in proteins can be site
for carbohydrate addition
(N-linked glycosylation)
BASIC AMINO ACIDS
Amino side chain of Lys and imino side chain of Arg have pK > 10
and carry a full +1 charge at neutral pH
Ring imino side chain of His has pK ~ 6.5
And carries on average a fractional positive charge at neutral pH
SULPHUR-CONTAINING AMINO ACIDS
Methionine is also considered
an aliphatic amino acid
Nearby cysteines on the
same or different
polypeptide chains
can undergo oxidation
to generate a covalent
DISULPHIDE bond
-
2e + 2H
+
PROLINE IMPARTS INFLEXIBILITY ON REGION OF POLYPEPTIDE
The propyl side chain of Proline is
covalently bonded to the beta-nitrogen
to form a ring.
Technically, proline is an IMINO acid
FREE
ROTATING
FREE
ROTATING
Beta-carbon at most amino acid residues
have TWO rotatable bonds
FREE
ROTATING
Beta-carbon at proline residues
has only ONE rotatable bond
AMINO ACID COMPOSITION AND SOLUTION pH
DETERMINE POLYPEPTIDE CHARGE
A protein’s
ISOELECTRIC POINT (pI)
is the pH at which the
protein’s NET CHARGE = 0
An “acidic protein” has
pI < 7
A “basic protein” has
pI > 7
A protein has
NET NEGATIVE CHARGE
when pH > pI
NET POSITIVE CHARGE
when pH < pI
pI is determined by
a protein’s amino acid
composition.
Different pIs of different polypeptides can be used to
separate these proteins by electrophoresis
at specific pHs.
Eg., an acidic protein has more
acidic residues than basic ones
NEXT LECTURE: PROTEIN STRUCTURE 1
Reading: Berg, Chapter 2