BCHE 7210 - Advanced Biochemistry II (Spring 2018)
Quiz I Study Guide
1. Standard Amino Acids: three-letter and one-letter codes
a. One-letter code notecards on Quizlet
2. Universal Structure of the amino acid (components)
a. Alpha carbon
b. NH3 group
c. COOH group (COO- when drawing)
d. R group (functional group)
e. Hydrogen atom
3. Properties of side chains
a. Notecards on Quizlet
4. Ionization states of amino acids; Zwitterion
a. Low pH = fully protonated (NH3+ and COOH)
i.
COOH becomes deprotonated around 2 pH
b. Neutral pH (7) → (NH3+ and COO-)
c. High pH = fully deprotonated (NH2 and COO-)
i.
NH3+ becomes deprontaed around 10 pH
d. Zwitterion: molecule with two or more functional groups, of which at least one has
a positive and one has a negative electrical charge and the net charge of the
entire molecule is zero
5. pK1, pK2, pKr and pI
a. Demonstrate the pH levels at which a functional group will deprotonated
b. pI = averages of pKa’s
i.
pH at which the molecule carries no electrical charge
6. Chiral Center
a. Alpha carbon connected to four DIFFERENT functional groups
7. Optical Rotation; Polarimeter
a. Chiral carbons have the ability to polarize light
b. Polarimeter: an instrument for measuring the polarization of light, and especially
(in chemical analysis) for determining the effect of a substance in rotating the
plane of polarization of light
i.
Clockwise → l
ii.
Counterclockwise → d
c. SEPARATE FROM ABSOLUTE CONFIGURATION
8. Enantiomers
a. Mirror images
b. Non-superimposable
9. Absolute Configuration of Amino Acids
a. D and L configuration
b. Amino acids normally have the L configuration
10. Fisher’s Convention: L and D Amino Acids
a. L-configuration used for this class → NH3+ on the LEFT side of the drawing
11. Corn Law
a. COOH → R group → NH3+
b. Helps dictate absolute configuration (L or D)
12. Peptide bond vs. Peptide Unit
a. Peptide bond - bonds between the carboxyl group and an amino group
i.
Carbonyl group to an amino end
ii.
Loss of water - condensation reaction
b. Peptide unit
i.
N-terminus → C-terminus of a single amino acid (together form peptides)
ii.
Peptide bond + phi angle + psi angle
c. Peptide group
i.
Rigid planar strucutre
ii.
Phi = psi = 180 (all in the trans configuration)
13. Peptides
a. Dipeptide = two peptides → 20^2
b. Tripeptide = three peptides → 20^3
c. Polypeptides = multiple peptides → 20^20
14. Amino Acid Classification
a. Hydrophobic-aliphatic
b. Hydrophobic-aromatic
c. Neutral-polar side chains
d. Acidic
e. Basic
15. Amino Acid vs. Imino acid
a. Amino acid - primary; only one carbon bonded to the amine nitrogen
b. Imino acid - secondary; proline → two carbons bonded to the amine nitrogen
i.
No flexibility
16. Hydrogen Bonding
a. Intra- or intermolecular interactions between an electronegative atom and a
hydrogen atom
i.
Alpha helices - intramolecular hydrogen bonding
ii.
Beta sheets - intermolecular hydrogen bonding
17. Hydrophobic interactions
a. Occur between non-polar groups
i.
The nonpolar characteristic is what drives certain amino acids the the
INSIDE of a protein
b. Avoid interaction with water
18. Polar Structure of of the Peptide Bond
a. There is a distribution of charge across the peptide bond due to resonance
b. Carbonyl oxygen has a slightly negative charge
c. The amino group has a slightly positive charge
d. Therefore, the peptide bond/back bone is POLAR
e. **Hydrogen bonding along a peptide chain → decreases the polarity of peptide
bonds → pushes to the inside of proteins (hydrophobic)
19. Resonance Hybrid Structure of Peptide Bond
a.
20. Torsion angle/rotation angle
a. Limitations on the angles outside of the peptide bond
b. Phi bond = N → C-alpha
c. Psi bond = C-alpha → COOH
21. Ramachandran Diagram
a. Demonstrates the sterically allowed conformations of phi and psi bonds
22. Cis vs. Trans Conformation of the Peptide Bond
a. Cis - same side of the peptide bond
b. Trans - opposite sides of the peptide bond
23. Levels of Structure
a. Primary: amino acid sequences
b. Secondary: primarily alpha helices and beta sheets; 3D arrangement
c. Tertiary: domains and motifs
d. Quaternary: multimeric proteins; multiple proteins interacting with each other
24. Helix
a. P = helical pitch
b. N = number of repeating units per turn
c. D = helical rise per repeating unit (p/n)
25. Alpha Helix: intramolecular hydrogen bonding
a. Right-handed: appears to be counterclockwise; positive N value
b. Left-handed: appears to be clockwise; negative N value
26. Beta-pleated Sheet
a. Two strands with intermolecular hydrogen bonding
b. Antiparallel: running in opposite directions
i.
More frequent due to stability
ii.
Hydrogen bonds are shorter - direct positioning
iii.
One amino acid interacting with one amino acid
c. Parallel: running in the same direction
i.
Hydrogen bonds are longer
ii.
One amino acid interacting with two amino acids
27. Terminus
a. N-terminus: amino group side of peptide chain
b. C-terminus: COOH side of a peptide chain
c. Polypeptide chains run from the N-terminus to the C-terminus
28. Disulfide bond
a. Two thiol groups bond together to form a disulfide bond
b. Occurs between Cysteine amino acids on
29. Reverse turns: a reverse turn is region of the polypeptide having a hydrogen bond from
one main chain carbonyl oxygen to the main chain N-H group 3 residues along the chain
a. Type I
b. Type II
30. Coil conformations
a. Keratin: two stranded coiled coil - two strands with similar stretches of repeats
come together and coil around each other → left handed coil
i.
Residues a and d are predominately nonpolar
ii.
Hydrophobic parts outside come together to form aggregate
iii.
Not soluble, tightly packed
b. Coil: loop conformation
i.
Alpha helix connected to a beta-pleated sheet
c. Fibroin structure: beta sheet structure
i.
Every second amino acid is a glycine
ii.
Every other amino acid is a serine or alanine
iii.
Forms a zipper-like structure - strong non-stretchable
d. Random coil: polymer conformation where the monomer subunits are oriented
randomly while still being bonded to adjacent units.
i.
It is not one specific shape, but a statistical distribution of shapes for all
the chains in a population of macromolecules.
31. Motifs: supersecondary structure
a. Do not have any functions; unstable; cannot usually fold by themselves
b. Helix-turn-helix: composed of two α helices joined by a short strand of amino
acids and is found in many proteins that regulate gene expression.
c. Hairpin B motif: a simple protein structural motif involving two beta strands that
look like a hairpin
i.
Consists of two strands that are adjacent in primary structure
ii.
Oriented in an antiparallel direction
iii.
Linked by a short loop of two to five amino acids
d. B - a - B motif: parallel beta-strands are connected by longer regions of chain
which cross the beta-sheet and frequently contain alpha-helical segments
32. Protein domain
a. Conserved part of a given protein sequence and (tertiary) structure that can evolve,
function, and exist independently of the rest of the protein chain
i.
Each domain forms a compact three-dimensional structure and often can be
independently stable and folded
33. Protein Secondary Structure: The Prediction
a. Basic idea - amino acid residues have preferences for certain secondary structures
i.
Prediction becomes an issue of pattern classification
34. Ampholytes: amphoteric molecules that contain both acidic and basic groups and will
exist mostly as zwitterions in a certain range of pH
35. Polyampholytes: Polyelectrolytes that bear both cationic and anionic repeat groups
36. Polyelectrolytes: a polymer that has several ionizable groups along the molecule,
especially any of those used for coagulating and flocculating particles during water
treatment or for making electrophoretic gels
37. Enzymatic activity
38. Characteristics of an Assay
a. Specific
b. Rapid
c. Sensitive
d. Quantitative
39. Fractionation Steps
a. Cell lysis→ centrifugation of cell debris (dispose of pellet/keep supernatant) →
b. Soluble fraction → precipitation of nucleic acids from supernatant →
c. Protein fraction → crude fractionation →
d. Enriched protein fraction → liquid chromatography →
e. Purified protein
40. Unit of Enzymatic Activity
a. 1.0 unit of enzyme activity is defined as the amount of enzyme causing
transformation of 1.0 umol of substrate per minute at 25 degrees Celsius under
optimal conditions of measurement
41. Activity vs. Specific Activity
a. Activity: total units of enzyme in a preparation
b. Specific activity: number of enzyme units per milligram of total protein
42. Protein Solubility and pH
a. Repulsion can occur at super high or low pH
b. At the isoelectric point:
i.
Can precipitate protein
ii.
Solubility is the lowest
43. Small Ions and Macroion Interactions
a. A protein is a macroion
b. Placing a macroion is a salt solution → ion cloud around the protein
i.
Ion cloud keeps proteins from interacting
c. No salt = proteins aggregate
i.
Resuspend in a buffer with salt
44. Salting In and Out
a. Salting in: at low concentrations, adding salt will increase the solubility/hinder
aggregation because the salt creates an ion cloud around the protein
b. Salting out: at high concentrations, salt will decrease solubility of the proteins
i.
Salt will uses water → proteins precipitate out
c. Low salt = protein soluble
d. High salt = protein insoluble
e. No salt - protein insoluble
45. ELISA: Enzyme-Linked Immunosorbent Assay
a. Sandwich
i.
Antibody in well
ii.
Test antigen added - if complementary, binding occurs
iii.
Secondary enzyme-linked antibody is added
iv.
Binds to test antigen = sandwich
b. Indirect
i.
Antigen in well
ii.
Test serum with antibody added - if complementary, binding occurs
iii.
Secondary enzyme-linked antibody is added
iv.
Binds to test antibody
46. Equilibrium Density Gradient Centrifugation - fractionation of proteins based on density,
independent of size using sucrose concentrations
a. Create a centrifuge gradient with varying sucrose concentration
i.
Low density on top
ii.
Intermediate density in the middle
iii.
High density on bottom
b. Suspended protein in the column
c. Spin column to separate protein by density in comparison to the sucrose
concentration layers
d. Collect each layer and test for protein of interest
47. Chromatography
a. Ion-exchange: separation based on the reversible electrostatic interaction of
proteins with the separation mixture
i.
Positively charged protein binds to negatively charged beads
ii.
Negatively charged protein binds to positively charged beads
iii.
pH-based
1. Must have a buffer ions bound to matrix during equilibrium to be
replaced by proteins
2. pH determines the charge on proteins
a. Above = negative
b. Below = positive
3. Protein of interest binds to beads
iv.
Salt-based
1. Increasing salt concentration will release proteins from beads due
to a salt concentration gradient
v.
Exchanges
1. Anion: DEAE
2. Cation: CM
vi.
**No volume restriction - capacity is a problem
b. Gel Filtration = Size-exclusion
i.
Pour mixture into column to pass through gel filtration resin
ii.
Large molecules cannot interact and pass first
iii.
Small molecules interact with the ‘sponge’ and are passed later
iv.
**Not good for large amounts
1. Can only load 5% of volume of column
c. Affinity Chromatography
i.
Circular DNA + plasmid into cell
ii.
Use a restriction enzyme to insert gene into plasmid
1. A histidine tail is added to the gene to ‘tag’ it
iii.
Histidine tail binds to Nickel-Sepharose Bead
48. SDS-PAGE
a. Process used to separate proteins based on their mass to charge ratio
b. SDS denatures proteins and gives them negative charges equally
c. The polyacrylamide gel contains pores of different size
d. Small proteins travel faster than larger
e. The negatively charged proteins will move toward the positive electrode upon the
application of an electrical current
49. Molecular Weight and SDS-PAGE
a. Separation of protein based on ratio of MW to charge
b. Not completely sure of this process
c. Plot distance travelled vs log MW to determine MW of unknown protein
d. Use amino acid sequence and MW of amino acid to calculate MW of protein
50. Roles of beta-mercaptoethanol and DTT in SDS-PAGE
a. BM: reducing agent to denature protein disulfide bonds
b. DTT: reducing agent to denature protein disulfide bonds
51. Isoelectric Focusing
a. Amino acids are incorporated into a gel creating a pH gradient
b. Protein solution is added
c. Electric field is applied
d. Proteins distribute along pH gradient according to pI
52. Chemical Modification vs. processing
a. Modification: adding a chemical group
b. Processing: protein trimmed to proper size
53. Acetylation of Proteins
a. Most common
b. Protects from degradation
c. N-terminus
d. Modification
54. Fatty Acid Acylation
a. Protect from degradation
b. Anchors protein to membrane
c. N-terminus
55. Modified Amino Acids
56. Roles of Protein Phosphorylation/Glycosylation/Methylation
a. Phosphorylation/Dephosphorylation: regulates cell processes (active vs. inactive)
b. Glycosylation: affects the 3D configuration of proteins
i.
Determines function and stability
c. Methylation: can inhibit or promote protein-protein interactions
57. Proteolytic cleavage: enzymes can catalyze the hydrolysis of peptide bonds between
amino acids (proteases, peptidases)
a. Insulin
i.
Initially → preproinsulin
ii.
Cut to proinsulin via signal peptidase
iii.
Insulin
b. Chymotrypsin
i.
Cleaves on the C-terminus of Phe, Trp, Tyr
58. Protein Self-Splicing/intein/extein
a. Intein: segment of protein that is able to excise itself
b. Extein: remaining portions of protein after an intein has removed itself
i.
Peptide bond
59. Ubiquitination: addition of ubiquitin to a protein → mark for degradation, alter location,
affect activity, promote/prevent protein interactions
a. E1: activating enzyme of ubiquitin → one
i.
ii.
iii.
Receives ubiquitin by forming a thioester linkage with Cysteine
Transfers ubiquitin to E2
Energy is released by ATP hydrolysis to (AMP and PPI)
b. E2: conjugating enzyme → few
i.
ii.
Receives ubiquitin from E1 by forming a thioester linkage with Cysteine
Trans Thiolation reaction
c. E3: protein ligase → many
i.
Ubiquitin is transferred to a Lysine on target protein forming a covalent
bond
ii.
Multiple classes
60. Isopeptide linkage: amide bond that is not present on the MAIN chain of the protein
a. Between a carboxyl terminus of one protein and the the amino group of a Lysine
of another
i.
Glycine of ubiquitin → Lysine of target protein
61. Proteasome
a. Structure: multisubunit enzyme
i.
19S
ii.
Core: two beta/two alpha rings
b. Function: degrades proteins tagged by ubiquitin
i.
19S cap binds to ubiquitin chain
ii.
Denatures protein due to hydrophobic conditions
iii.
Feeds protein into barrel core
iv.
Protein is degraded
v.
Ubiquitin is recycled via isopeptidase
62. Edman degradation
a. PITC: reagent that allows for the progressive removal and identification of the Nterminal amino acid in an acidic environment
b. Steps:
i.
PITC + N-terminus of peptide
1. pH 8 at 45C
ii.
PTC-peptide
1. Anhydrous TFA gas/liquid
iii.
ATZ derivatives(unstable) + (N-1) peptide
1. H+/H2O
iv.
PTC-amino acid
v.
PTH-amino acid
63. Invariant amino acid: conserved; essential for structure and/or function of the protein
a. Cannot be replaced
64. Variable amino acid: not conserved; (considerable) variation of the amino acid within the
primary structure of a protein
65. Conservative Amino Acid Substitution: an amino acid is exchanged for another that has
similar properties (basic, acid, nonpolar, etc.)
66. Protein Sequences and Phylogenetic Relationships
a. Calculate the number of amino acids that differ between species to determine
how closely they are related
b. Shows common ancestry by showing species that share common ancestors
c. Rates of evolution may vary
i.
Histones: slow
ii.
Hemoglobin: fast