(OLD) (Dr. Choy) (Unit A) Topic 3: Protein Structure and Analysis Flashcards
Proteins are…
Linear polymers of amino acids
**
Define:
Dipeptide
Peptide chain with two amino acids
Define:
Tripeptide
Peptide chain with 3 amino acids
Define:
Oligopeptide
Peptide chain with 3-20 amino acids
Define:
Polypeptide
Peptide chain with many residues
Define:
Protein
1 or more polypeptide chains
True or False:
Peptide bonds exhibits resonance
True
What results from a peptide bond’s resonance?
40% double bond causes planar structure (cannot rotate around bond)
What bonds are rotateable in a peptide chain? What are they known as?
- N-Cα, known as Φ rotation angle
- Cα-C, known as Ψ rotation angle
List:
Levels of Protein Structure
- Primary
- Secondary
- Tertiary
- Quarternary
Define:
Primary protein structure
Amino acid sequence of protein (residues)
Define:
Secondary protein structures
Spatial arrangement of polypeptide backbone
Define:
Tertiary protein structure
3-D structure of entire polypeptide, including side chain(s) (only 1 subunit)
Define:
Quaternary protein structure
Spatial arrangement of polypeptide chains in protein with multiple subunits
What are the two major types of secondary structures?
- α-helices
- β-sheets
True or False:
Some part of proteins do not have secondary structures
True
What are secondary structures stabilized by?
Usually stabilized by H-bonds
* Between backbone N-H and C=O groups
What are hydrogen bonds? When are they the strongest?
- Hydrogen interaction with electronegative atoms
- When in a straight line
A hydrogen bond is —- the strength of a covalent bond in an aqueous solution
1/20
Describe:
α-helices
- ~3.6 residues per turn
- 5.4 angstroms rise along axis per turn
- C=O forms H-bond with N-H group 4 residues down the strand
Why are there not many prolines or glycines in α-helices?
- Proline causes kinks to appear in helices
- Glycine destabilizes the helix (has many conformations, not favourable for helix)
What forms β-sheets?
Hydrogen bonds betwen neighbouring strands
What are the two types of β-sheets?
- Antiparallel sheets (N to C beside C to N)
- Parallel sheets (N to C beside N to C)
What bonds form β-sheets?
Hydrogen bonds between the carboxyl and amide groups
State:
Classes of Protein Structures
- All-α protein
- All-β protein
- α/β protein
- Intrinsically disordered protein
What does an “intrinsically disordered protein” mean?
Means that the protein has no significant amount of secondary structure
Tertiary structures involve the spatial arrangement of…
- Chain’s regular - Irregular secondary structures
- Conformations of side chains
What is the 3D structure of a protein stabilized by?
Interactions between side chains and backbone atoms
True or False:
Interactions in tertiary structures can be between resides distant in the sequence
True
List:
Intermolecular interactions by strength (strongest to weakest)
(in aqueous solution)
- Covalent
- Ionic
- Hydrogen
- Van der Waals force
What types of van der Waals forces are there?
- Between permanent dipoles
- Dipole induced dipole interactions
- London Dispersion Forces
What other interactions are possible in tertiary structure?
- Ionic interactions (between charged amino acids)
- Disulfide bonds
Describe:
Disulfide bonds
Strong bonds, formed between chains
* Specifically between cysteine molecules
* Stabilizes protein
* Broken by strong reducing agent
* Only occurs in oxidizing environment
What types of representations of proteins are there?
- Backbone model
- Ribbon model
- Wire model
- Space filling model
- Electrostatic potential map
What are quaternary structures stabilized by?
- Hydrogen bonds
- Ionic bonds
- Van der Waals interations
- Disulfide bonds
- Others
What type of hydrogen bonds can occur in quaternary structures?
- Backbone to backbone
- Backbone to sidechain
- Sidechain to sidechain
Why are quaternary structures important?
Have strong implications in protein function (ex. hemoglobin)
Define:
Hydrophobic effect
When proteins fold:
* Hydrophobic side chains mainly fold into core
* Polar and charged side chains are on the outside
Why must the hydrophobic effect occur?
- Water forms extensive Hydrogen bonds and have high level of freedom
- Hydrophobic groups in solution decreases bonds between water and decreases entropy (unfavourable)
Describe:
Entropy changes in protein folding
- Entropy of water increases
- Entropy of protein decreases
What are the function of chaperone proteins?
Helps proteins to form properly
What type of chaperone proteins are there?
- Directly binding ones that can guide protein into correct conformation
- Chamber-like with a cap, allows protein to properly fold inside the chaperone protein
What forms of protein denaturation are there?
- Chemical denaturation
- Thermal denaturation
Define:
Chemical denaturation
- Add chaotropic agents (eg. urea)
- Increases solubility of nonpolar substance in water
- Can refold protein by removing chemical
Define:
Thermal Denaturation
- Proteins unfold by applying heat
E.x. Albumin in eggs
* Proteins unfold (linearized)
* Forms random bonds
* Forms disulfide bonds
* IRREVERSIBLE
What are protein domains?
Distinct region of protein
* Can fold independently from each other
* Provide structure/function (or both)
* Only TERTIARY (one subunit)
Why is protein purification necessary?
Purify protein samples to study a specific protein function
* Get rid of impurities, inhibitors etc.
What are the 3 types of chromatography?
- Size Exclusion Chromatography
- Ion Exchange Chromatography
- Affinity Chromatography
Describe:
Size Exclusion Chromatography
Uses porous gel beads (has a gel matrix inside)
* Smaller proteins go into bead (slower to go through)
* Larger proteins go around bead (faster to go through)
Does shape of protein affect the speed in size exclusion chromatography?
Yes
Describe:
Ion Exchange Chromatography
Uses charged beads (positive charged ones in anion exchange columns)
* Negatively charged proteins bind to beads
* Uncharged and positively charged proteins go through
* Increase salt concentration to get remainder “stuck” proteins to detach
Can ion exchange chromatography be done with negatively charged beads?
Yes, it would be called cation exchange column (collecting positively charged proteins)
Define:
Isoelectric point
The point (pH) where the charge of the protein is 0
What would happen if:
1. pH > pI
2. pH < pI
- The protein’s overall charge is negative
- The protein’s overall charge is positive
Describe:
Affinity Chromatography
Uses beads containing a ligand that can only bind to a specific protein
* Those proteins will get stuck with the ligand
* The other proteins are washed down with the buffer
Define:
SDS-PAGE
Analytical technique
* SDS = Sodium dodecyl sulfate
* PAGE = Polyacrylamide Gel Electrophoresis
What is sodium dodecyl sulfate in SDS-PAGE?
- Ionic detergent
- Binds to hydrophobic groups
- Helps solubilize them
- Average 1 SDS binds to 2 proteins
- Results in overall positive charge complex
What does SDS-PAGE show?
An gel apparatus with negative charge at the bottom and positive charge at the top
* Smaller proteins move down to the negative end faster
* Larger proteins get stuck in the gel and move slow
What is mass spectroscopy used for?
Used to characterize proteins in mixtures
* Isolate proteins via isolation
* Fragmentation
* Mass analysis
What information does mass spectroscopy provide?
- Sequence
- Abundance
- Various modifications in protein
What does X-Ray Crystallography tell us? What are its limitations?
Shows us 3D structures of the protein
* Protein must be able to crystallize
* Needs to be done with pure samples
What is NMR? What is its benefits?
Nuclear Magnetic Resonance
* Shows 3D structures of small proteins
* Shows ensemble of structures
* Good for small proteins
* No need for crystallization
