Biochemistry- Chapter 2 & 3 Mississippi State University

Water has a higher melting point, boiling point, and heat of vaporization than

most other common solvents

Hydrogen bond

electrostatic attraction between the oxygen atom of one water molecule and the hydrogen of another.

Hydrogen bonds are

relatively weak.
- bond dissociation energy = ~23 kJ/mol in liquid H2O
- 10% covalent, 90% electrostatic.

Hydrogen bonds are fleeting

- lifetime of each hydrogen bond is just 1 to 20 picoseconds in liquid
- when one hydrogen bond breaks, another forms.
- Flickering Clusters

Hydrogen bonds readily form between

an electronegative atom and a hydrogen atom covalently bonded to another electronegative atom

electronegative atom

the hydrogen acceptor.

hydrogen atom covalently bonded to another electronegative atom

the hydrogen donor

Hydrogen atoms covalently bonded to carbon atoms

DO NOT do hydrogen bond

Alcohols, aldehydes, ketones, and compounds containing N—H bonds all form

hydrogen bonds with water.

directionality of the Hydrogen Bond

strongest when the acceptor atom is in line with the covalent bond between the donor atom and H
- maximizes electrostatic interaction

Hydrophilic

describes compounds that dissolve easily in H2O; generally charged or polar compounds.

Hydrophobic

nonpolar molecules such as lipids and waxes.

Amphipathic

contain regions that are polar (or charged) and regions that are nonpolar.

H2O dissolves

salts and charged biomolecules by screening electrostatic interactions.

The increase in entropy of the system is largely responsible for

the ease of dissolving salts in water.

water has a high

dielectric constant

Dielectric constant (ε)

a dimensionless physical property that reflects the number of dipoles in a solvent - for water at 25 °C, ε = 78.5 - for nonpolar benzene at 25 °C, ε = 4.6

the force (F) of ionic interactions in solution depends on:

- the magnitude of the charges (Q)
- the distance between the charged groups (r)
- the dielectric constant of the solvent (ε)

ionic attractions or repulsions operate

over 10 to 40 nm.

CO2, O2, N2

are nonpolar.

Why is CO2, O2, N2 non polar?

Their movement into aqueous solution decreases entropy by constraining their motion.

Nonpolar compounds interfere with the hydrogen bonding among H2O molecules by

increases enthalpy (∆H) and decreases entropy (∆S)

the free-energy change (∆G = ∆H −T∆S) for dissolving a nonpolar solute in water is unfavorable because

- ∆H has a positive value
- ∆S has a negative value
- ∆G has a positive value

H2O molecules form a highly ordered, cage like shell around each solute molecule

- maximizes solvent-solvent hydrogen bonding
- H2O molecules are not as highly oriented as those in clathrates (crystalline compounds of nonpolar solutes and water)

Hydrophobic effect

- nonpolar regions cluster together
- polar regions arrange to maximize interactions with each other and with the solvent

micelles

thermodynamically stable structures of amphipathic compounds in water.

van der Waals interactions (London dispersion forces)

distance-dependent weak attractions and repulsions between transient dipoles.

van der Waals radius

measure of how close an atom will allow another to approach.

Noncovalent interactions are

much weaker than covalent bonds, Continually forming and breaking.

For macromolecules, the most stable structure

usually maximizes weak interactions.

In every living organism

proteins are constructed from a common set of 20 amino acids.

amino acids share common structural features

-α carbon and four substituents - α
carbon is the chiral center
• Tetrahedral
• Exception: Glycine has a second hydrogen atom instead of an R group

Two possible stereoisomers

enantiomers

D and L system specifies

absolute configuration.

L-amino acids are

used by the cells to make proteins. (NH3 on left.)

amino acids form

zwitterions

In neutral pH (pH=7)

both alpha amino and carboxyl group are ionized.

The charged form of the amino acid is called a

zwitterion.

Amino groups, Carboxyl groups, and ionizable R groups =

weak acids and bases.

Zwitterion

occurs at neutral pH.

pKa

analogous to pH and defined by the equation:

pKa = log 1/Ka= −log Ka

The stronger the tendency to dissociate a proton

the stronger the acid and the lower its pKa.

All amino acids have

at least two dissociation constants.

Several Amino acids have an ionizable side chain with a

third pKa

The pH at which the net electric charge is zero is the

isoelectric point (pI)

For amino acids without ionizable side chains, the Isoelectric Point(equivalence point, pI) is:

At this pint, the net charge is zero.

Henderson-Hasselbalch equation

describes the shape of the titration curve of any weak acid.
-Can be used to determine the fraction of ionizable groups found in each of the possible ionization states at a given pH.

Amino acids can be sorted into four groups on the basis of the general characteristics of their R groups:

- Hydrophobic amino acids
- Polar amino acids
- Positively charged amino acids
- Negatively charged amino acids

pKa of amino acids depends on

the neighboring molecules in a protein.

essential amino acids

histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine

non-essential amino acids

alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine

Polar Amino Acids Have Side Chains That Contain an

electronegative atom

Histidine is found at the active sites of many enzymes that require a

proton donor or proton acceptor.

Alpha-amino acid pKa

9.30

Arg pKa

12.48

Lys pKa

10.53

Tyr pKa

10.07

Cys pKa

8.18

His pKa

6.00

Glu

4.25

Asp

3.65

Alpha-Carboxyl

2.10

Amino acid side chains play a pivotal role in

protein folding and protein-protein interaction.
- These side chains may become ionized at different pKa values.
-This has an effect on overall protein structure.

If the side chain does not have an ionizable group

then the pI is simply the average of the α-NH3 and α-COOH pKa values.

If the side chain has an ionizable group then

all three pKa values must be considered.

If the side chain is acidic (asp and glu), then

average the sidechain pKa with the α-COOH pKa

If the side chain is basic (his, arg, and lys), then

average the sidechain pKa with the α-NH3 pKa.

For other ionizable groups (tyr and cys), determine which is the middle pKa

average it with the α-COOH pKa.

When the pH > pI

a protein has a net negative charge.

When the pH < pI,

a protein has a net positive charge.

Proteins can be separated and purified based on

size
- charge
- binding properties
- protein solubility

Methods for Purifying Proteins

1. first step = break open tissue or microbial cells. (crude extract= releases proteins in solution.)
2. second step = fractionation = separate proteins into fractions based on size or charge- "salting out" = lower solubility of proteins in salt to selectively precipitate proteins
3.third step = dialysis = use semipermeable membrane to separate proteins from small solutes

column chromatography steps:

first step = buffered solution (mobile phase) migrates through porous solid material (solid phase)
2. second step = buffered solution containing protein migrates through solid phase• protein properties affect migration rates

Ion-Exchange Chromatography

separates based on sign and magnitude of the net electric charge.
-pH and concentration of free salt ions affect protein affinity
-Recall the relation between pH and pI
-uses bound charged groups:
-- cation exchangers
-- anion exchangers

pH=pI

no net charge

pH>pI

negative charge

pH

net positive charge

Size-Exclusion Chromatography

--also called gel filtration chromatography --separates based on size
--large proteins emerge from the column before small proteins do

Affinity Chromatography

--separates based on binding affinity
--eluted by high concentration of salt or ligand

Amino acid sequence can inform:

- 3D structure
- function
- cellular location
-evolution

consensus sequence

reflects most common amino acid at each position

Bioinformatics:

- identifies functional segments in new proteins.
--establishes sequence and structural relationships to known proteins

Essential amino acid residues =

conserved over evolutionary time.

Less important amino acid residues =

vary over evolutionary time.

Horizontal gene transfer =

transfer of a gene or group of genes from one organism to another.
-- proteins derived from transferred genes are not good candidates for bacterial evolution studies.
-- for example, rapid spread of antibiotic-resistance genes in bacterial populations

Homologs = homologous proteins =

members of protein families.
--identified by comparing protein sequences to a database of protein sequences.

paralogs

homologs in same species

orthologs

homologs in different species.