BIOL710: Molecular BiologyMondays and Wednesdays, 5:30PM

LECTURERS

Mitchell GoldfarbHN834 772-5289Goldfarb@genectr.hunter.cuny.edu

Thomas Schmidt-GlenewinkelHN841 772-5027Thomas@genectr.hunter.cuny.edu

COURSE WEBSITE (for Goldfarb lectures)

http://biology.hunter.cuny.edu/Goldfarb/

Then click link to course at bottom of the web page

SUGGESTED TEXT

Biochemistry, 6th editionBerg, Tymockzo, & Stryer

ISBN: 0716787245OR

Biochemistry, 4th editionStryer

ISBN: 0716720094

Available online fromwww.barnesandnoble.com

www.amazon.com

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

Exams and Studying

Midterm on Oct. 22nd (tentative)Covers Lectures 1-12 on Proteins (Goldfarb and Schmidt-Glnewinkel)

Final in DecemberCovers Lectures 13-26 on DNA/RNA & Information Flow (Schmidt-Glenewinkel)

Study Tips

Read suggested readings before and/or soon after each lecture

Distribute your studying throughout semester; do not cram

LECTURE 1: pH, pK, Amino Acids, Peptide Properties, pIReading: 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

Hydrogen

Carbon

Oxygen

Nitrogen

Sulphur

Phosphorus

H

C

O

N

S

P

ElectropositiveForms 1 covalent bond

Forms 4 covalent bonds

Strongly electronegativeForms 2 covalent bonds

Weakly electronegativeForms 3 covalent bonds

Weakly electronegativeForms 2 covalent bonds

In oxidized formForms 5 covalent bonds

CH

H

C

H

H

H

O H

CH

H

C

H

O

O

H

ethanol

acetic acid

CN

C

C

H

O

O

H

H

H

glycine

CN

H

C

H

O

O

H

H

H

HH

S Hcysteine

R -- O -- P -- O -- H

O

O- phosphate side group

Oxidation and Reduction of Organic Compounds and Oxygen

OXIDATION -- Removal of an electron pair from a moleculeREDUCTION -- 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

CH

H

C

H

H

H

O H

CH

H

C

H

H

O2 H+2 e +

-

ETHANOL (reduced) ACETALDEHYDE (oxidized)

O2 + 2NADH + 2H+ 2H2O + 2NAD+

O

O

OH H

ACCEPTOR DONOR REDUCED 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.

CH

H

C

H

O

O

H CH

H

C

H

O

O

-

H+

CO

H

C

H

H

N

H

H H

H

H

+

H+

CO

H

C

H

H

N

H

H

H

H

Conjugate ACID Conjugate BASE

acetic acid acetate

2-amino-ethanolhydroxyethylammonium

pH

Water is in equilibrium with its minor dissociation products, H+ and OH-

While 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] = 7Acidic 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.

A strong BASE is an METAL HYDROXIDE compound that FULLY DISSOCIATES into

OH- and its positive counterion in water.

pH of strong acid and base solutions

HYDROCHLORIC ACID NITRIC ACID

HCl H+ Cl- HNO3 NO3-H+

SODIUM HYDROXIDE CALCIUM HYDROXIDE

NaOH Na+OH- Ca(OH)2 Ca+2 2OH-

pH = - log10 [ACID] pH = 14 + log10 [BASE]

pH of 10 mM HCl = 2 pH of 0.1 M NaOH = 13

e.g. e.g.

TITRATION OF A STRONG ACID SOLUTION WITH STRONG BASE

14

0

0

2

2

1

1

54

3

876

1312

11109

NEUTRAL

pH

Molar Equivalents of NaOH

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

pKa of an acid/base conjugate pair

HA H+ + A-Acid Proton Base

Ka = [H+ ][A- ]

[HA]

TAKE LOGARHYTHMMULTIPLY BY -1 pH = pKa + log10

[A-][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 BaseThe weak acid BUFFERsBUFFERs 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

0

0

2

1

1

0.5

5

4

3

7

6

Molar Equivalents of NaOH

pH

pH

For acetic acidpKa = 4.0

For unbuffered0.1 M acetic acid

pH = 2.5WHY???

pKa

25

75

100

50

[HA

] (mM

)[H

A] (m

M)

[A[A--] (m

M)

] (mM

)

pH of a Weak Acid Solution in Water

HA H+ + A-

pH = pKa + log10

[A-][HA]

For a weak acid in water: [A-] = [H+] and [HA] = [A]total

= pKa + log10 [H+] - log10 [A]total

Therefore:

pH = (pKa + log10 [A]total)

2

Three Types of Non-Covalent Bonding InfluenceIntramolecular and Intermolecular Interactions

ELECTROSTATIC BONDING

HYDROGEN BONDING

Oppositely charged groups are attractive.

R

C O

O- R

H

H

HN +

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.

R O H O

C

R

N H

H

O

H

R

OH

H

OH H

O

H

H

Three Types of Non-Covalent Bonding InfluenceIntramolecular 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 ~ 3.0pK of amino group is ~ 9.5

POLYPEPTIDES

FREEROTATING

FREEROTATING

All polypeptides synthesizedfrom L-amino acids

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

AROMATIC AMINO ACIDS

NOTE:

Tyrosine isalso a

hydroxylatedaminoacid

NOTE:

Methionine isalso a

sulphur-bearing

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

AMIDO AMINO ACIDS

Carboxylic acid side chain in Asp and Gluhas pKa ~ 4.5 and carries

a full -1 charge at neutral pH

Asn is amidated version of AspGln is amidated version of Gln

Asn and Gln are NOT charged,but are higly polar

NH2 group on Gln in proteins can be sitefor carbohydrate addition(N-linked glycosylation)

BASIC AMINO ACIDS

Amino side chain of Lys and imino side chain of Arg have pK > 10and carry a full +1 charge at neutral pH

Ring imino side chain of His has pK ~ 6.5And carries on average a fractional positive charge at neutral pH

SULPHUR-CONTAINING AMINO ACIDS

Methionine is also consideredan aliphatic amino acid

2 H+2 e +

-Nearby cysteines on the

same or differentpolypeptide chains

can undergo oxidationto generate a covalent

DISULPHIDE bond

FREEROTATING

FREEROTATING

PROLINE IMPARTS INFLEXIBILITY ON REGION OF POLYPEPTIDE

The propyl side chain of Proline iscovalently bonded to the beta-nitrogen

to form a ring.

Technically, proline is an IMINO acid

Alpha-carbon at most amino acid residueshave TWO rotatable bonds

Alpha-carbon at proline residueshas only ONE rotatable bond

FREEROTATING

AMINO ACID COMPOSITION AND SOLUTION pH DETERMINE POLYPEPTIDE CHARGE

A protein’sISOELECTRIC POINT (pI)is the pH at which the

protein’s NET CHARGE = 0

An “acidic protein” haspI < 7

A “basic protein” haspI > 7

A protein hasNET NEGATIVE CHARGE

when pH > pI

NET POSITIVE CHARGEwhen pH < pI

pI is determined bya protein’s amino acid

composition.

Eg., an acidic protein has moreacidic residues than basic ones

Different pIs of different polypeptides can be used toseparate these proteins by electrophoresis

at specific pHs.

NEXT LECTURE: PROTEIN STRUCTURE 1Reading: Berg, Chapter 2