CHEM 237: Primary Primary Structure, Purification and Sequencing

What reaction are peptides made from?

condensation of amino acids

Proteomics

study of the structure and function of proteins in the human body

What are the 2 Protein Sequencing Techniques?

Gene & Chemical/Enzymatic

What does the gene sequence tell you?

protein sequence, size, charge, structure and function

Can the protein function tell you anything?

not really; use separation techniques

What does separation of proteins rely on?

physico-chemical properties

What are examples of the Physico-Chemical Properties that contribute to protein seperation?

size

charge

affinity to a ligand

solubility

hydrophobicity

thermal stability

What kind of molecules is compatible with UV absorbance?

aromatic structures

What does Separation based on Solubility rely on?

ionic strength

Ionic strength

measure of the total concentration of ions in solution

What is the relationship between concentration and ionic strength?

As concentration increases, ionic strength increases

How does pH affect solubility separation?

As pH increases, solubility decreases

What will proteins that's pH=pI do?

precipitate; loss of charge-charge repulsion

How does thermal stability affect solubility?

As proteins unfold, they precipitate

What techniques separate proteins based on size?

chromatography (gel filtration & size exclusion)

Elution Profile

visual representation of elution peaks; the concentration of a substance that leaves the column

What does Elution Profile say about Elution time?

Largest molecules elute first

Smallest molecules elute last

What is pI?

isolectric point

What does isoelectric point determine?

the pH where the protein has no net charge

What is the charge of protein if pH < pI?

positive

What is the charge of protein is pH > pI?

negative

How is isoelectric point determined experimentally?

IEF (Isoelectric Focusing)

How does IEF work?

a sample is placed in a pH gradient gel and subjected to an electric field, causing the molecule to migrate until it reaches the pH point where its net charge is zero

What determines the charge of protein?

the buffers pI & pH

What resin will negatively charged proteins bind to?

positive charged resin (anion exchange resin)

What resin will positively charged proteins bind to?

negative charged resin (cation exchange resin)

What are examples of positively charged resin (anion exchange resin)?

DEAE sepharose, Q sepharose, bead-linker quaternary amine

What are examples of negatively charged resin (cation exchange resin)?

CM & SP

What should you change to get the protein off the resin after seperation?

pH & salt concentration

What molecule will come off the elution in a cation exchange column first?

weakly bound +, lower pI

What is the protein charge if pI < pH?

negative charge

What is the protein charge if pI > pH?

positive charge

Affinity

the specific and strong attraction

ATP columns

purify proteins that bind to ATP

Ni Columns

bind metal or His proteins

What kind of proteins will bind to ATP columns?

proteins that bind to ATP

How to get the protein off ATP columns?

add ATP

What does Polyacrylamide gel electrophoresis (PAGE) separate based on?

separates proteins based on size

How are proteins prepared for PAGE?

boiled in SDS containing DTT to denature it and break disulfide bonds

What is the purpose of boiling proteins?

separate multi subunit proteins

What charge are the proteins in SDS?

NEGATIVE (regardless of amino acid composition since they are coated in SDS)

What does SDS stand for?

sodium dodecyl sulfate (detergent)

What controls the rate of movement in SDS?

size (small = faster, large = slower)

Why do we want to separate proteins?

characterize the protein, sequence, structure

Primary Structure

sequence of amino acids in a chain

Secondary Structure

helix or pleated sheet

Tertiary Structure

Results from interactions between side chains.

Quaternary Structure

4 separate chains assembled into an oligomeric protein

What method can we use on all proteins for sequence determination?

Quantitative Amino Acid Analysis

What does & doesn't Quantitative Amino Acid Analysis provide analysis on?

Does: amino acids present in a protein

Doesn't: sequence information

Steps of Quantitative Amino Acid Analysis Procedure

1. Hydrolyze AMIDE bonds

2. Ion exchange column (with cation exchange at low pH)

What is DABS used for?

change a compound's structure to improve its analysis of hydrolyzed amino acids

What does End-Group Analysis allow you to Identify?

the amino acids present at the N-terminal and C-terminal of a protein & the number of polypeptide chains in the protein

What do N-terminal reagents react with?

free amino groups

What does detecting 2 or more amino acids indicate?

heterogeneity OR polypeptide chain

What type of labelled amino acids can standards identify?

DNP or Dansyl labelled amino acids

What scenario would disulfide bonds need to be cleaved?

cystine residues can interfere with sequence determination procedures

What is the process called that Cleaves Disulfide Bonds?

disulfide exchange

What reagents will break disulfide bonds?

B-mercaptoethanol or DTT (dithiothreitol)

What structure will DTT (dithiothreitol) form after disulfide cleavage?

cyclic disulfide

What combination of reagents will prevent disulfide bonds from reforming?

carboxymethylation & iodoacetate

What length of amino acid chains will sequencing be reliable for?

a chain of 30-50 amino acids

What reagents are used to make smaller peptides?

chymotrypsin and trypsin

What is the first step in the protein and peptide sequencing process?

Reduce disulfide bonds

What is the second step in the protein and peptide sequencing process?

Cleave polypeptide to smaller fragments

What is the third step in the protein and peptide sequencing process?

Sequence fragments

What is the fourth step in the protein and peptide sequencing process?

Overlap sequences to determine overall sequence

What is the fifth step in the protein and peptide sequencing process?

Repeat without reduction of disulfides to locate position of bridges

What reagents are used for Enzymatic Cleavage Procedures?

Trypsin and Chymotrypsin (cleave to create new N-term and C-term)

What reagent is used fro Chemical Cleavage Procedures?

Cyanogen Bromide (cleaves at the methionine)

What does Cyanogen Bromide generate after cleavage?

lactone

What is the Edman Degradation Method?

used to detect N-terminal amino acid sequence

What is the one requirement that amino acids must undergo prior to Edman Degradation?

reduce disulfide bonds to Cys

What is the overall process to sequence a protein?

1. Purify protein

2. Break disulfide bonds (DTT or B-Mer.)

3. Break peptides (trypsin or chymotrypsin)

4. Separate peptides by ion exchange or HPLC

5. Detect AA sequence at N-term via Edman Degradation Method

6. Analyze the data to generate the intact peptide

How are long proteins sequenced?

1. Isolate the gene

2. Use DNA sequencing to sequence the DNA

3. Translate the DNA sequence into a protein sequence

Mass Spectrometry

a electrospray ionization that separates proteins according to their mass

What charge does trypsin generate after cleaving a protein into peptides?

positive (+) charge

What is the purpose of Peptide Mapping?

analyze the primary structure of a protein by breaking it down into smaller peptide fragments through enzymatic digestion

What amino acids does Trypsin cleave at?

Lys (K) & Arg (R)

(unless next to Pro (P))

What amino acids does Chymotrypsin cleave at?

Phenylalanine (F), Tryptophan (W), and Theronine (Y)

What amino acids does Cyanogen Bromide (CNBr) cleave at?

Methionine (M)

What does Edman Degradation identify?

the first amino acid of the peptide from N-term.

What's Mass Spectrometry and ESI?

a technique used to measure the mass-to-charge ratio

How does Mass Spectrometry Work?

1. Ionization

2. Mass Analyzer

3. Detection

4. Fragmentation

What's ESI?

a method used to ionize large molecules

What's Tandem Mass Spectrometry?

Identifies proteins; sequences peptides by fragmenting them and analyzing

How to Calculate Protein Mass from ESI?

1. Obtain the m/z value (peaks corresponding to the m/z ratio)

2. Formula: Mass = m/z value

3. Protein mass (peak with the highest intensity = used to estimate the protein's most likely mass)

What are the Pros and Cons of Amino Acid Analysis?

Pro: works on large proteins

Con: no protein sequence, no modification detection, slow speed

What are the Pros and Cons of Edman Degradation?

Pro: provides sequence

Con: doesn't work on large proteins, no modification detection, slow speed

What are the Pros and Cons of Mass Spectrometry?

Pro: provides sequence, works on large proteins, detects modifications, fast speed

What are the 5 drawbacks of 6N HCl hydrolysis? (IS THIS RELEVANT?)

1. Destruction of certain amino acids (Trp, Cys, Asn & Gln)

2. Time-Dependent Degradation (prolonged hydrolysis)

3. Racemization (Ser & Thr converts into D-amino acids)

4.Loss of Sequence Information

5. Formation of Side Products

How would you get a protein bound to a column and off the column?

increase pH

or

increase salt concentration

What's "Salting Out"?

Adding excess salt decreases protein solubility, causing precipitation

Removes water, promotes aggregation

What's "Salting In"?

Adding a small amount of salt increases protein solubility

Shields charges, prevents aggregation