Review topics

Exam1 Review Topics

September 22, 2015Introduction

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, P2. Covalent and non-covalent bonds in biomolecules

Covalent Proteins

Peptide bond

Nucleic Acid

Phosphodiester bond

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

R = 8.314 J/mol K

G = H TS

At equilibrium:

G = 0; S = H/T

Keq = [C][D]/[A][B]

G = -RT ln Keq Free energy change for non-standard state concentrations:

G = G + RT ln [C][D]/[A][B]

EX:

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

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 Cysteine2. 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 Phenylalanine (Phe, F)F Isoleucine (Ile, I)

I Leucine (Leu, L)

L Methionine (Met, M)

M Tryptophan (Trp, W)

W Alanine (Ala, A)

A Valine (Val, V)

V Proline (Pro, P)

P POLAR Cysteine (Cys, C)

C Tyrosine (Tyr, Y)

Y Glycine (Gly, G)

G Glutamine (Gln, Q)

Q Asparagine (Asn, N)

N Serine (Ser, S)

S Threonine (Thr, T)

T ACIDIC (neg) Aspartic Acid (Asp, D)D Glutamic Acid (Glu, E)E BASIC (pos) Arginine (Arg, R)

R Histidine (His, H)

H Lysine (Lys, K)

K Aromatic Tryptophan

UV Abs. @ 280 (HIGHEST)

Tyrosine

UV Abs. @ 270

Phenylalanine

UV Abs. @ 260 (LOWEST)

Cysteine is the ONLY amino acid capable of forming disulfide bonds3. 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 Acidic

Aspartic Acid Glutamic Acid

Histidine

Basic

Arginine Cysteine

Lysine

Serine Threonine

Tyrosine

Glycine: 2 pKa values

Glu, His, Lys: 3 pKa values5. Isoelectric point of amino acids: Gly, Glu, His, Lys

Glycine: pI = 5.97 Glutamic Acid: pI = 3.22 Histidine: pI = 7.59 Lysine: pI = 9.946. The peptide bond

A carbonyl-OH and an amino-H form a H2O to bring Two amino acids together, connecting C-N

Double bond shared b/w C=O and C=N 40% double bond character

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 Resins containing positively charged groups attract NEG charged solute

If pH > pI: Protein = NEG charged; Use anion exchange

EX: DEAE cellulose (weakly basic POS)

Cation exchange resins

Resins containing negatively charged groups attract POS charged solute If pH < pI; Protein = POS charged; Use cation exchange

EX: CM cellulose (weakly acidic NEG)

2. Gel filtration chromatography; principles and applications

Separate molecules by SIZE LARGER molecules excluded from the gel beads

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 GST fusion proteins ( use Glutathione Sepharose 4B resin

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

Elute with Glutathione ( pure GST fusion proteins

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 A stable pH gradient (HIGH to LOW pH) established after applying electric field to the gel Add protein and reapply electric field; then staining

SDS-PAGE: Separation by Molecular Weight 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 Cleaves C-side of Arg & Lys

Cyanogen Bromide (CNBr)

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

Predicted (-helical structure of protein (1951) John Kendrew

X-ray diffraction of myoglobin

Electron density map of protein

Max Perutz

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: ( = 180 and ( = 0

( = 0 and ( = 180

3. Structural features of the (-helix

Average peptide dihedral angles ( = -57 and ( = -47

Number of hydrogen bonds

# H-bonds = n-4

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 Causes kinks hinders helix formation

4. Structural features of the (-sheets

Exists in parallel and anti-parallel form

Parallel:

( = -120 and ( = 105

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 4 per heavy chain 2 per light chain Intersubunit disulfide bonds 2 hold the heavy chains together 1 holds each heavy chain to a light chainTertiary and Quaternary structures of proteins

1. Recognize protein folds: (, (, (/(, (+( (

(-helices dominate the structure

( (-sheets are the primary feature

(/( (-helices and (-sheets mixed within a domain (+( (-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

? dafuq(-helices

(-sheets