Exam1 Review Topics

September 22, 2015

Introduction
1. Modular structure of biomolecules; building blocks of proteins, nucleic acids, cellulose
and starch
Proteins: C, H, O, N, S

Carbohydrates: C, H, O, (N) (CH2O)n

DNA and RNA: C, H, O, N, P

Lipids:C,H,O,N,P

2. Covalent and non-covalent bonds in biomolecules

Covalent
o Proteins
Peptide bond
o Nucleic Acid
Phosphodiester bond
o Carbohydrates
Glycosidic bond
Non-covalent

3. Principles of thermodynamics; G, Go, Keq, and related calculations

The standard state is normally unit activity simplified to 1 M concentration
Denoted by superscript degree sign
o
R = 8.314 J/mol K
G = H TS
At equilibrium:
G = 0; S = H/T
o

Keq = [C][D]/[A][B]
G = -RT ln Keq
o
Free energy change for non-standard state concentrations:
G = G + RT ln [C][D]/[A][B]
o
EX:
o

Water and pH
1. The hydrogen bond; hydrogen bonds in proteins
Bent angle polar
H-bonds occur in highly Eneg atoms
Directional & Saturable
2. The definition of pH; calculation of pH, [H+] and [OH-]
pH = -log10[H+]
10-pH = [H+]
EX:
3. Acid and base theory; conjugate acid and base
A strong acid has a conjugate weak base
A strong base has a conjugate weak acid
4. The definition of pKa and experimental meaning of pKa
pKa defines the acidity of an H+ atom in a solution
pH = pKa when a solution contains 0.5 eq acid & 0.5 eq base
o When there are equal concentrations of acid/base
5. pH calculation using Henderson-Hasselbach equation
pH = pKa + log10 [A-]/[HA]
EX:
6. Buffers; optimum pH of buffer solutions; calculate pH of buffer solutions
pH = pKa + log10 [A-]/[HA]

Optimum pH: when pH = pKa

Use ICE tables to calculate change in pH of buffers
EX:

7. Buffer systems of cells; the titration curve of H3PO4 and related calculation

EX:
Amino acids
1. Stereochemistry of amino acid: L-amino acids are building blocks of proteins
All amino acids are chiral (except Glycine whose side chain is H)
Most naturally occurring amino acids are S, with exception of Cysteine
2. Properties of amino acid: aliphatic, polar, negatively charged, positively charged,
aromatic, disulfide bond and cysteine; UV absorption by aromatic amino acids; average
molecular mass of amino acid residues in proteins (110 Da/residue)
NONPOLAR
o Phenylalanine (Phe, F)
F
o Isoleucine (Ile, I)
I
o Leucine (Leu, L)
L
o Methionine (Met, M)
M
o Tryptophan (Trp, W)
W
o Alanine (Ala, A)
A
o Valine (Val, V)
V
o Proline (Pro, P)
P
POLAR
o Cysteine (Cys, C)
C
o Tyrosine (Tyr, Y)
Y
o Glycine (Gly, G)
G
o Glutamine (Gln, Q)
Q
o Asparagine (Asn, N)
N
o Serine (Ser, S)
S
o Threonine (Thr, T)
T
ACIDIC (neg)

o Aspartic Acid (Asp, D)

D
o Glutamic Acid (Glu, E)
E
BASIC (pos)
o Arginine (Arg, R)
R
o Histidine (His, H)
H
o Lysine (Lys, K)
K
Aromatic
o Tryptophan
UV Abs. @ 280
(HIGHEST)
o Tyrosine
UV Abs. @ 270
o Phenylalanine
UV Abs. @ 260
(LOWEST)
Cysteine is the ONLY amino acid capable of forming disulfide bonds

3. Structure of the 20 amino acids; one letter and three letter abbreviations of amino acids
4. Acid base properties of amino acids; titration curve of Gly, Glu, His, Lys
Amino acids are weak polyprotic acids

R-group pKa

o Acidic
Aspartic Acid
Glutamic Acid
Histidine
o Basic
Arginine
Cysteine
Lysine
Serine
Threonine
Tyrosine
Glycine: 2 pKa values

Glu, His, Lys: 3 pKa values

5. Isoelectric point of amino acids: Gly, Glu, His, Lys

Glycine: pI = 5.97
Glutamic Acid: pI = 3.22
Histidine: pI = 7.59
Lysine: pI = 9.94
6. The peptide bond
A carbonyl-OH and an amino-H form a H2O to bring
o Two amino acids together, connecting C-N
o Double bond shared b/w C=O and C=N
o 40% double bond character

o Trans-conformation
7. Draw structures of short peptides;
identify amino acids and naming of short peptides.
Named from N-terminal to C-terminal
Ser-Gly-Tyr-Ala-Leu OR SGYAL

Protein purification
1. Ion exchange chromatography; principles and applications
Separate molecules by CHARGE by exchanging
ion of interest with salt ion
Increasing salt concentration until protein washed out
Anion exchange resins
o Resins containing positively charged groups
attract NEG charged solute
o If pH > pI: Protein = NEG charged; Use anion exchange
o EX: DEAE cellulose (weakly basic POS)

Cation exchange resins

o Resins containing negatively charged groups attract POS charged solute
o If pH < pI; Protein = POS charged; Use cation exchange
o EX: CM cellulose (weakly acidic NEG)

2. Gel filtration chromatography; principles and applications

Separate molecules by SIZE
LARGER molecules excluded from the gel beads
o Emerge from the column sooner than SMALLER molecules
3. Affinity chromatography: GST fusion and His tag protein purification (how to, what
kind of resins to use: Glutathione Sepharose and Ni-NTA Sepharose; how to elute the
protein from these resins: Glutathione and imidazole)
Separate molecules by BINDING SPECIFICITY
Exploits the biological function of the target protein binding to certain ligands
Protein immobilized by binding to specific resin
o GST fusion proteins use Glutathione Sepharose 4B resin
o 6xHis tag protein use Ni-NTA Sepharose resin

Eliminate unwanted contaminating proteins by washing and filtration

Elute desired protein by adding high concentrations of the free ligand
o Elute with Glutathione pure GST fusion proteins
o Elute with imidazole pure 6xHis tag proteins
4. Protein analysis by SDS-PAGE; measuring molecular weight and purity of proteins
Separates proteins by MOLECULAR WEIGHT
SMALLER molecular weight = HIGHER mobility (moves farther distance)
Estimate MW by comparing to Molecular standards (Mr)
EX:
5. Isoelectric focusing and two-dimensional gels (isoelectric focusing and SDS-PAGE,
proteins are separated based on their pI and MW)
Isoelectric focusing: Separation by pI
o A stable pH gradient (HIGH to LOW pH) established after applying
electric field to the gel
o Add protein and reapply electric field; then staining
SDS-PAGE: Separation by Molecular Weight
o Molecules of lower MW travel farther
Primary structure of proteins
1. Techniques to derive amino acid sequences of proteins: chemical sequencing by
Edman degradation and mass spectrometry sequencing
Analysis of N-terminal
Only good for short peptides (50 amino acids)
2. Specificity of proteases trypsin (cleave after Lys or Arg) and cyanogen bromide
(CNBr, cleave after Met)
Trypsin
o Cleaves C-side of Arg & Lys
Cyanogen Bromide (CNBr)
o Cleaves C-side of Met homolactone Serine
3. Assembly of polypeptide sequence from fragment sequences
EX:
Secondary structure of proteins
1. The contribution of Linus Pauling, John Kendrew and Max Perutz in understanding the
structure of proteins
Linus Pauling
o Predicted -helical structure of protein (1951)
John Kendrew
o X-ray diffraction of myoglobin
o Electron density map of protein

Max Perutz
o Structure of hemoglobin
2. Planar structure of the peptide group; definition of the dihedral angles and
(phi) = The angle about the C N bond
(psi) = The angle about the C Co (C=O) bond
Planar structure forbids these angles because of steric hindrance:
o = 180 and = 0
o = 0 and = 180

3. Structural features of the -helix

Average peptide dihedral angles
o = -57 and = -47
Number of hydrogen bonds
o # H-bonds = n-4
o EX: 26 amino acid residues 22 H-bonds
3.6 residues per turn
5.4 per turn
1.5 per residue
Right handed (clockwise)
RARELY contain Gly and Pro
o Causes kinks hinders helix formation
4. Structural features of the -sheets
Exists in parallel and anti-parallel form
o Parallel:
= -120 and = 105
o Anti-parallel:
= -135 and = 140
Each single strand has 2 residues per turn
The hydrogen bonds are interstrand
Ribbon representation
Porin structure
5. Ramachandran plot and dihedral angles of -helix and -sheets

Immunoglobulins

Composed of two heavy chains (53-75 kD)

and two light chains (23 kD)
Intrasubunit disulfide bonds
o 4 per heavy chain
o 2 per light chain
Intersubunit disulfide bonds
o 2 hold the heavy chains together
o 1 holds each heavy chain to a light chain
Tertiary and Quaternary structures of proteins

-helices

1. Recognize protein folds: , , /, +

o -helices dominate the structure

-sheets

o -sheets are the primary feature

o -helices and -sheets mixed within a domain

+
o -helices and -sheets are separated to some extent

2. Understand the basic principles of experimental methods to determine protein

structures: X-ray crystallography and NMR spectroscopy
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