Question 1:

Match each of the following techniques with the property or properties that the SEPARATION is based upon.  If another property is involved but is less important, put it in parentheses.  (You may use A - E more than once and you may give more than one answer per blank, but give just the best one or two for each blank).

SDS-PAGE                             _____                          A.  Size

Size Exclusion                         _____                          B.  Shape

Salting out                               _____                          C.  Charge

Isoelectric  focusing                _____                          D.  Specific binding

Affinity column                      _____                          E.  Solubility 

DEAE Column                       _____

Ultracentrifugation                 _____

Question 2: In a recent paper, an alanine to valine substitution in a protein is described which is associated with a higher risk of heart disease.  Discuss briefly two different reasons an alanine to valine mutation might be harmful.  (Hint:  2° and 3°)

Question 3: Which of the following will reduce a disulfide containing protein?

a)         iodoacetic acid                        d)         guanidine hydrochloride (GuHCl)

b)         DTT                                         e)         mercaptoethanol

c)         high temperature                     f)         performic acid

Question 4:  An over-the-counter hair care product sometimes called "activator" is used to relax curly hair.  What type of compound might be the active ingredient?

Gelatin desert products (like Jello brand gelatin) are actually made from melted down collagen from horse connective tissue.  Name three amino acids or modified amino acids that are probably present in fairly high concentration in gelatin.

Question 5. Matching   (one answer per blank)

_____  Fibrous protein consisting of three strands of 3₁₀ helix.                    A.  b-keratin

_____  Fibrous protein found in feathers.                                                     B.  a-keratin

_____  Fibrous protein found in hair and also in hooves, nails.                    C.  silk

_____  Fibrous protein with beta-sheets with many Ala, Gly residues         D.  collagen

Question 6: Insulin is a polypeptide hormone that contains two short polypeptide chains linked by two interstrand disulfide bonds. The most logical order of events to perform in order to sequence this protein would be

A: The peptides are reduced with mercaptoethanol.

B: The peptides are sequenced using Edman chemistry.

C: The peptides are separated by chromatography techniques.

D: The peptides are alkylated with iodoacetamide.

Question 7: Reaction of the peptide, ala-met-lys-ser, with phenylisothiocyanate (PITC) at pH 8.0 followed by mild acidification (first cycle of Edman chemistry):

a. would release the labeled peptide PTH-ala-met-lys-ser

b. would release PTH-ala, PTH-ser, PTH-lys, and PTH-ser

c. would release PTH-ser and the peptide ala-met-lys

d. would release PTH-ala and the peptide met-lys-ser

e. none of the above.

Question 8: Homologous proteins do NOT:  (Circle two best answers)

a. have identical lengths.

b. have identical N-terminal and C-terminal amino acids.

c. share a significant degree of sequence similarity.

d. perform the same function in different organisms.

e. have sequence identity in direct correlation to the relatedness of the species from which they were derived.

Question 9: A Ramachandran plot shows:

a. the amino acid residues which have the greatest degree of rotational freedom.

b. the sterically allowed rotational angles between R groups and carbons in a peptide.

c. the sterically allowed rotational angles between C and the amide nitrogen (C -N) as well as between C and the amide carbonyl carbon (C -CO).

d. the sterically allowed rotational angles about the amide nitrogen (NH) and CO.

e. the amino acid residues that form helix, sheet , etc.

Question 10: The repeating secondary structures as found in -strands, -turns, and helices of folded proteins primarily result from:

a. hydrogen bonds.
b. electrostatic interactions.
c. hydrophobic interactions.
d. van der Waals interactions.
e. ionic interactions.

Question 11: If the following section of a polypeptide is folded into an -helix, to which amino acid is   the carbonyl group of alanine hydrogen bonded?

                 ala-ser-val-asp-glu-leu-gly

a. serine

b. aspartic acid

c. glutamic acid

d. leucine

e. valine

Question 12: An amino acid commonly found at the end of an helix (helix-breaking behavior) is:

a. phe (F).

b. leu (L).

c. glu (E).

d. met (M).

e. pro (P).

Question 13: Polylysine is a random coil when the pH is less than 11, while it forms an -helix if the pH is raised to greater than 12. This is because at pH 12 :

a. the lysine residues are negatively charged which electrostatically stabilizes the helix.

b. the positive charges on the lysine residues stabilizes the -helix.

c. the lysine residues are neutral which eliminates electrostatic repulsion between the R groups.

d. the high concentration of OH^- ions in solution reduces the electrostatic repulsion between the R-groups.

e. the lysine side chain changes configuration with pH.

Question 14: The amino acid residue most likely to be found in a beta turn is:

a. glycine.

b. alanine.

c. valine.

d. glutamic acid.

e. leucine.

Question 15: Interpret the following observations in simple terms of size, shape, etc:

A. Protein X has an apparent molecular mass of 180 kD by size exclusion chromatography but an apparent molecular mass of 61 kD by SDS-PAGE. What is the likely reason for this?

B. If 2-mercaptoethanol is added and the solution heated before loading the SDS-PAGE gel, Protein X has an apparent molecular mass of 32 kD. Give your interpretation of data and Sketch Protein X both before and after treatment with 2-mercaptoethanol.

Be creative and use different shapes to show the structure and label types of bonds or NCIs.