Primary Structure Analysis

Question 1

What is a protein domain?

Answer 1

A conserved part of a given protein sequence and structure that has a specific function

Question 2

How many AA domains have?

Answer 2

10-500 amino acids

Question 3

What does AA analysis estimate and help with

Answer 3

Estimates nutritional value of food and solubility of proteins
Gives better understanding of protein structure and function
Peptide broken down to amino acid via heat and HCl

Question 4

What does protein sequencing compare and predict?

Answer 4

Compares between normal and mutant proteins; similar proteins in different species
Predicts 3D protein structure to design drugs

Question 5

Features of Edman sequencing

Answer 5

Is a continuous cycle
Works best for small polypeptides
Can have many possible sequences of cut fragments

Question 6

What are the cleavage agents in Edman sequencing?

Answer 6

CNBR: Met (M) residues
Trypsin: Lys (K) and Arg (R) C-termini
Chymotrypsin: Phe (F) Tyr (Y) Trp (W) C- termini

Question 7

What are the cleavage agents used for in Edman sequencing?

Answer 7

Peptide mapping
When sequencing large proteins, at least 2 deavage agents are needed to yield more manageable overlapping fragments

Question 8

What is the reagent of Edman sequencing?

Answer 8

PITC phenylisothiocyanate

Question 9

Process of Edman sequencing

Answer 9

1. Purify protein to homogeneity
2. Cleave all disulfide bonds to fully unfold
3. PITC reacts with N-terminal AA under alkali conditions; acid added n-terminal released as pth- derivative
   4.PITC reacts with new n-terminal
4. Cycle repeats

Question 10

What does mass spec sequencing compare?

Answer 10

Ion fragments data with computer database of known protein structures

Question 11

How does the mass spec separate molecules?

Answer 11

Fragments are ionized and separated based on their mass:charge ratio (accelerate along chamber path at rate proportional to their mass)

Question 12

Sample preparation for protein purification

Answer 12

1.homogenize tissue in a suitable buffer
2.protein mixture fractioned into cell fractions (via centrifuge)
3.fraction dialized into suitable buffer compatible with chromatography

Question 13

Describe gel filtration chromatography

Answer 13

Based on molecule size differences
Bigger molecules make it down the gel column faster- don’t fit in gel pores

Question 14

Describe ion-exchange chromatography

Answer 14

Separates molecules based on their charge at a certain ph
Reversible absorption of charged solute molecules to immobilized groups of opposite charge

Question 15

Describe affinity chromatography

Answer 15

Separates based on ligand interactions
Targets protein that interact with the ligand, nonspecific protein passes through

Question 16

Describe his-tag proteins and Ni chromatography

Answer 16

DNA encoding protein contains a his-tag primer that attaches during PCR after ligation in a vector

Question 17

When is protein purification successful?

Answer 17

If total protein reduces faster than total activity