Biochem Test 2

Oxidoreductases

transfer of H, O, or e-

Transferases

Transfer of a functional group

Hydrolases

Formation of 2 products from 1 by adding H2O

Lyases

Breaking a bond without adding H2O

Precipitation

• Add (NHs)2SO4 until you get your target protein at the bottom as a precipitate (pellet)

• As you increase salt concentrations, proteins precipitate out at a characteristic concentration

Column Chromatography- Stationary Phase

Nonmoving part of the column

Column Chromatography- Mobile Phase

Solution that moves through the column and can carry sample

Column Chromatography- Detection

Method for seeing if protein is present in flow through- often 280 nM absorbance

Column Chromatography- Fraction

Part of total solution that has come through column

Ion-Exchange Chromatography

Separates compounds based on charge

Ion-Exchange Chromatography- Stationary Phase

Charged positive or negative resin

Ion-Exchange Chromatography - Mobile Phase

Buffer with varied ionic strength

• often just higher [NaCl] to increase ionic strength and pull samples off resin

Cation Exchange Chromatography (CM)

• binds cations (+)

• Resin had (-)

Anion exchange chromatography (DEAE)

• binds anions (-)

• Resin had a (+)

For Ion-Exchange Chromatography (anion/cation) what is the relationship between pH and pI

pI=pH, if the molecule is not attracted to the stationary phase then it comes right off. For example, if its anion (-) and the charge is a positive charge then it comes off and vice versa for cation (+) and a negative charge.

Affinity Chromatography

• Separates compounds based on interactions with other compounds

Affinity Chromatography- Stationary Phase

Polymer resin with attached interaction partners (ligands)

Affinity Chromatography - Mobile Phase

Buffer and then buffer with interacting partner

• Often increases concentration of ligand in solution

Size Exclusion Chromatography

Separates compounds based on size in their nature, bigger compounds come out earlier

Size Exclusion Chromatography- Stationary phase

Polymer resin with small compounds channels/pores to “trap” smaller compounds

• normally effective over specified ranges (10,000-100,000 Da aka 1 gram per mole)

Size Exclusion Chromatography- Mobile Phase

Buffer

• doesn’t change during experiment

General Protein Detection

• Light absorbance at 280 nM (from aromatic acids)

• Bradford Assay

Bradford Assay is what kind of protein detection and what does it do?

General protein detection, it dye binds roughly even to all proteins, dye alone is brown, dye and protein is blue, and more protein dyes brighter blue

Specific Protein Detection

• Antibodies bind to specific molecules (like proteins)

• Allow for identifying specific protein quantity

Gel Electrophoresis

separates compounds based on charge, shape, and size

• Smaller proteins move faster

• Problem: some proteins are positive and some are negative

• Less bands means it is more pure and one protein is identified

• More bands means more proteins were identified and less pure

SDS Electrophoresis

• Denaturing gels use detergents to mostly get rid of charge dependence

  • most common detergent: SDS

• Takes proteins and unfolds them/denatures them

  • allows protein to be separated by size but you lose information about the protein

Isoelectric Focusing

Proteins applied on a pH gradient stop moving when they have no charge (aka pH=pI)

If pH is GREATER than pI what is the charge

negative

If pH is LESS than pI what is the charge

Positive

Relationship between electrophoresis and isoelectric focusing

If you start with isoelectric focusing you can find the 0 charge (pH = pI) and then you can take that and do gel electrophoresis to find information about the protein

What is the combination of isoelectric focusing and gel electrophoresis called and how does it work?

2-D Electrophoresis, you look at pI in accordance with pH strip (nonpolar amino acids have neutral pI and charged ones have a low pI) and then you look at the size (smaller things move down the gel quicker)

Sanger Sequencing

• Problem: proteins can contain 100s of amino acids

• Solution: break up big polypeptides into smaller ones

• 1) Label N-Terminus with FDNB or Dansyl Chloride or Dabsyl Chloride

• 2) Determine total amino acid content (% of each amino acid)

• 3) Cleave into smaller peptides using proteases or other reagent

• 4) Sequence them

• 5) Put them back together

For Sanger Sequencing if you’re given a peptide what do you do?

Look at sanger reagent result and cut it at N-Terminus and see what amino acid is present (this goes first), check what digest is given and where that cleaves (this goes next), and then add on the last sequence

Primary Structure

order of amino acids (peptide bonds)

Secondary Structure

alpha helixes or beta sheets (hydrogen bonding)

Tertiary Structure

Secondary structure elements fold interacting with one another

• R group interactions - hydrogen bonding, ion-ion, disulfides (covalent), hydrophobic effects

Quaternary Structure

Interaction of multiple folded polypeptide chains (driven by all interactions types)

• Amino acids that are polar would be on the outside in solvent and nonpolar ones on the inside

Alpha Helix

• Right handed turn typically

• 3.6 amino acids per turn

• Each turn is 5.4 A long

• Optimizes peptide to peptide hydrogen bonding

• Amino acids are 4 apart and can interact with R-group interactions (they congregate on same side, proline and glycine are not favorable)

Alpha helixes and charges

• N-terminus has a slight positive charge

• C-terminus has a slight negative charge

• Favorable for R-groups with charge opposite dipoles to be at ends

How do you know what peptides form an alpha helix?

Look at every four amino acids and seeing which has charges alternating

Beta Sheet Confirmation

• Made of planes in a zig-zag pattern

• Stacks are stabilized by peptide hydrogen bonds between sheets

Fibrous Proteins

• Have alpha helix or beta confirmation

• structure is rod-like

• insoluble in water

• structural proteins in cells

Globular Proteins

• more compact structures

• hemoglobin

• include most enzymes and regulatory proteins

Motif

recognizable folding pattern involving two or more elements of secondary structure

Domain

Part of polypeptide chain that can undergo movement as a single entity

Intrinsically disordered proteins

Protein has no defined structure, and the change in structure determines its function

Protein Family

Proteins with significant similarity in primary structure and/or similar function and structure

Super Family

Two or more protein families that have little amino acid sequence overlap but have structural and/or function similarities

Ligand

A molecule that protein interacts with, can be any molecule type including a small molecule, another protein, DNA, sugar, and lipids

for P + L = PL, Ka =

[PL] / [P][L]

Fraction Occupied (Y or Theta)

The fraction of protein present that has ligand bound to it

Hyperbolic Curve

Rate of response (derivative) is higher at beginning and then levels off

1 / Ka =

Kd and is always at 0.5 on the Y

The smaller the Kd value

increase binding (affinity), the less ligand needed to get to 50% occupied

The higher the Kd value

the less ligand binding/affinity (weakest binding)

Myoglobin

• Other small molecule ligands can fit in the binding site

• The distal His is able to bind to hydrogen bond with oxygen but not carbon dioxide

• The structure helps maintain the function of binding oxygen

Oxygen binding ot myoglobin is what curve?

hyperbolic

Hemoglobin

• Myoglobin and a single subunit of hemoglobin are very similar

• Made up of four subunits

  • 2 alpha and 2 beta

  • each subunit is a separate polypeptide chain

T State of Hemoglobin

• Lower oxygen affinity (weaker O2 binding, larger Kd

• Stabilized by more ion pairs

• Tense

• Puckered heme state

• Drop in pH

• Inc in pCO2

R State of Hemoglobin

• Higher O2 affinity (higher binding, lower Kd)

• Relaxed

• Some ion pairs broken while few more are formed

• Heme is more planar

• Rise in pH

• Decrease in pCO2 and BPG

Oxygen binding curve for hemoglobin is what shape?

Sigmoidal

Hill Coefficient

slope = n

• max n is number of binding sites

• min n is 0 (no binding)

• n<1 means negative cooperatively

In the Hemoglobin Sigmoidal curve

• As we go left = tighter binding

• Lower pH = (+) charge = ion interact with Asp, T state, O2 unbinding, go right

• Higher pH = neutral charge = no ion-ion, R state, O binding, go left

• Inc BPG = T state, O2 unbinding, go right

Hemoglobin in lungs

• Higher pH

• Favors R state

• Binds O2

Hemoglobin in Tissues

• Lower pH

• T state

• Releases O2, aka O2 unbinding

If a reaction is releasing CO2

uncharged = no ion-ion = R state = O2 binding = go left

If a reaction yields a product with O-

allows for ion-ion = T state = O2 unbinding, go right

BPG

• Binding captivity is only present in T state

• Favors O2 unbinding

HbF

Has weaker affinity for BPG, allows fetuses to compete for O2 with parent

CoFactor

nonprotein component such as inorganic ion, complex organic, or metalloorganic

Coenzyme

a complex organic or metallorganic

Prosthetic Group

Cofactor that is strongly or even covalently bound

Holoenzyme

The “whole” structure, protein + cofactor

Apoenzyme

Just the protein part of the structure

Isomerases

Intramolecular rearrangement

Ligases

Join 2 reactants together by forming new bonds

• Intermediate (ES and EP)- stable state along reaction (minimum)

• Transition State (high peaks)- unstable, not populates state at barrier maximum \

• Δ𝐺rxn- energy difference between reactant and product state

• Δ𝐺 +- reaction barrier height, energy difference between transition state and reactants (speed)

The taller the barrier

the slower the reaction (rate limiting step)

General Acid-Base Catalysis

Involves proton transfer or abstraction that lowers transition state, catalyst does not change at the beginning or end, never has a covalent bond

Covalent Catalysis

Involves transient formation of enzyme-substrate covalent bond

Metal Ion Catalysis

Fe, Cu, Zn, Mn, Na, K, Ca