Exam 2: Chp 5 Flashcards
Describe the structure and function of fibrous proteins?
simple, linear structure, and insoluble in water; structural role
Describe the structure of globular proteins?
Spherical, hydrophobic amino acids inside, hydrophilic amino acids outside
Describe the structure and location of membrane proteins?
fewer hydrophilic amino acids, hydrophilic amino acids on outside of protein, and insoluble in water; embedded in a cell membrane
What is a protein primary structure?
the amino acid sequence
What is a protein secondary structure?
3D structures localized on part of the polypeptide chain; typically regular and repeating
What is a protein tertiary structure?
the whole 3D structure of one polypeptide chain formed via folding
What is a protein quaternary structure?
Arrangement of multiple folded polypeptide chains into one larger protein
What are three types of secondary protein structures?
⍺-helix
β-sheet
β-turn
What kind of interactions facilitate secondary, tertiary, and quaternary structure formation?
Noncovalent bonds
What kind of interactions facilitate primary structure formation?
Covalent bonds
What are three types of crude protein isolation/purification?
Centrifugation, salting out, or dialysis
How does centrifugation isolate/purify proteins?
Separates them based on density
How does salting out isolate/purify proteins?
Increasing salt concentration causes the salt to solvate solvate water ions, decreasing solubility of the protein
What are some limits of protein isolation/purification via salting out?
It only works to a certain point and its not efficient for separating a protein from a protein
How does dialysis isolate/purify proteins?
Diffusion across a semipermeable membrane separates proteins from small molecules
What is the definition of chromatography?
Solution of compounds is passed through a stationary phase
How does ion exchange chromatography purify/isolate proteins?
Separates proteins based on charge using a matrix with positively and negatively charged groups
How does size exclusion chromatography isolate/purify proteins?
Small proteins are slowed because they must travel through the inside of porous beads in a matrix; the large proteins travel between beads.
How does hydrophobic interaction chromatography isolate/purify proteins?
A matrix with hydrophobic groups that interact with non polar proteins, separating them from polar proteins
How does affinity chromatography isolate/purify proteins?
Specific ligand is bound to the protein of interest then covalently coupled to a matrix, all other compounds are washed away
What is high-performance liquid chromatography?
Any chromatography method with high molecular interactions made small scale, packed tightly, and ran with high pressure to push the protein through
What is a benefit of high-performance liquid chromatography?
produces a high resolution (good separation)
What are limits of high-performance liquid chromatography (HPLC)?
size and pressure
How does gel electrophoresis purify/isolate proteins?
An electric field propels proteins through an agarose or polyacrylamide gel that separates them based on size
How are proteins purified/isolated during SDS-PAGE electrophoresis
Based on size
What does SDS-PAGE stand for?
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
How does isoelectric focusing electrophoresis isolate/purify proteins
Proteins stop moving through pH gradient when their net charge reaches zero based on their relative amounts of acidic or basic groups
How does 2D electrophoresis isolate/purify proteins?
First isoelectric focusing is performed, then the gel matrix is rotated 90° and SDS-PAGE is performed
What is a purification scheme?
Several protein purification/isolation methods in sequence
What is the goal of a purification scheme?
to maximize specific activity
What is specific activity?
The ration of a protein of interest to total protein
What is of sacrifice of purification schemes?
material is lost during each step
What is acid hydrolysis?
The process of decomposing a polypeptide into individual amino acids using 6 M HCl and 110°C
What is the first step in determining primary structure (amino acid sequence) of a protein? (Step 1)
If quaternary structure exists then polypeptide chains should be separated and purified
How do you separate and purify polypeptide chains?
Disrupt their non-covalent interactions using pH, high salt, urea, or gaunidium chloride and isolate via chromatography
When determining protein primary structure; what is done after polypeptide chains are purified and separated? (Step 2)
Disulfide bonds are cleaved
What are two ways to cleave disulfide bonds?
1) Oxidation using performic acid
2) Reduction using 2-mercaptoethanol or dithrothrietol
When determining protein primary structure; what is done after disulfide bonds are cleaved? (Step 3)
Determine the N-terminal amino acid and the C-terminal amino acid
How is the N-terminal of a polypeptide chain determined?
Cleavage using an Edman reagent and identification of the PTH derivative
How is the C-terminal of a polypeptide chain determined?
Cleavage using carboxypeptidase
Why can carboxypeptidase or the Edman reagent not be used to determine long amino acid sequences?
Limited to 20-50 Ads because lost product builds up and muddles the reaction
When determining protein primary structure; what is done after the N-term and C-term AAs are determined? (Step 3)
The polypeptide is cleaved into smaller fragments and their sequences are determined using Edman or carboxypeptidase reactions
What are two ways polypeptides are cleaved into smaller fragments when determines AA sequences?
1) Enzymatically using a protease
2) Chemically
What proteases are used to enzymatically cleave a polypeptide during determination of primary structure?
Trypsin and Chymotrypsin
What end of the peptide bond is susceptible to cleavage by chymotrypsin? What amino acids?
C; Phe, Trp, and Tyr (rarely Leu)
What end of the peptide bond is susceptible to cleavage by trypsin? What amino acids?
C; Arg and Lys
What end of the peptide bond is susceptible to cleavage by staphylococcal protease? What amino acids?
C; Asp and Glu
What end of the peptide bond is susceptible to cleavage by cyanogen bromide? What amino acids?
C; Met
What is used to chemically cleave a polypeptide chain during primary structure determination? What does it do?
cyanobromide; cleaves the C-terminal side of Met and turns it into homoserine lactone
During primary structure determination; what is done after the initial cleavage and sequencing of a primary structure?
The polypeptide sequence is once again cleaved with a different method and the sequences are determined
During primary structure determination; what is done once the polypeptide sequence is cleaved and sequenced in too different ways?
The full sequence is constructed using overlapping fragments of the sequence fragments
What are homologous proteins?
Proteins with similar sequences found in different species that typically indicate evolutionary relatedness
What are post translational modifications? an example?
Covalent modifications after proteins synthesis that typically modify AA side chains; disulfide bonds
Which terminal does the peptide bond form on the amino acid containing the insoluble resin?
The N-terminal on the nucleophilic amine group
Which terminal does the peptide bond form on the amino acid containing the FMOC protecting group?
The activated C-terminal
What is used to activate the carboxyl of an amino acid that is adding to the N-terminal of another amino acid?
DIPCDI (diisopropylcarbodimide)
What is used to protect the amine group of an amino acid that is adding to the N-terminal of another amino acid?
FMOC-Cl (flourenylmethyloxylcarbonyl-Cl)
How is FMOC cleaved once a polypeptide is made?
Using weak organic base because it is base labile - piperidine
How is the insoluble residue removed once a polypeptide is made?
An acid, such as HF, because it is acid labile
How is a polypeptide purified from byproducts once it is made?
Filtered out because the byproducts are soluble and the insoluble resin keeps the polypeptide seperate
What is fp in terms of ligand and protein kinetics?
the fraction of protein that exists bound to ligand
What is a saturation curve?
ligand concentration on x-axis and fp on y axis
What is Kd in terms of ligand and protein kinetics?
A measure of binding affinity recording the concentration of ligand where 1/2 of protein has ligand bound
What does a smaller Kd mean?
the protein and ligand have a higher binding affinity
Function of 2-mercaptoethanol?
To cleave disulfide bonds
