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No regrade requests will be considered after this date.

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Class Average  40.5 points

Trypsin is an enzyme that cleaves amino acid chains immediately after positive residues, creating a new carboxyl terminus and a new amino terminus in the process.

(A) Give the specific name for the type of bond cleaved in these reactions. 

Peptide Bond

(B) Describe one structural characteristic of this bond.
(either of the below answer is acceptable)

Peptide bonds are rigid and planar. There is no rotation about the bond between the C and N atom.

Peptide bonds (1.33A) are shorter than typical C-N single bonds (1.46A) and longer than typical C=N double bonds (1.27A). This is due to electronic resonance of the O+=C-N structure.

(C) In the space below, draw the products resulting when the short amino acid chain E K V is treated with trypsin.  Show all atoms, including side chains.  Please be as neat as possible.

(D) In your drawing above, mark with an arrow any occurrences of the bond specified by Part (a).  Or, write “NONE” in the space below if no such bonds exist.
(E) Label each side chain with its full name and 3 letter abbreviation.

(A) Name at least two reasons why the phi and psi torsion angles of the polypeptide backbone of proteins need to be considered when looking at secondary structures. (4 points)

Need two of the following (2 pts each)

-steric hindrance of side chains.
-tells us about rotational freedom of side chain.
-tells about protein dynamics in aqueous environment.
-uniqueness of individual residue (because of specific angles)
-allows construction of Ramachandran plots.

(B) Complete the following sentence: The angle of rotation phi is around the N-alpha C bond whereas the angle of rotation psi is around the alpha C- carbonyl bond.  (2.0 points)

(C) Give two structural features that contribute to the stability of a coiled-coil. (4 points)

Need two of the following (2 pts each)

-"knobs in holes"
-H bonds between N and carbonyl
-hydrophobic effect
-Van der Waals interactions
-disulfide bridges

Below is the schematic representation of the enzyme carboxypeptidase.

B.  What motif (s) is (are) involved?  (3.0 points)
1.0  beta hairpin
B. beta-alpha-beta
1.0 beta-loop-beta
(mixed beta sheet is acceptable for one answer)

C. Draw the topological diagram, including N and C termini. (3.0 points) 

D. How many active sites are there? Label with * on the schematic diagram.  (1.5 points)
B. 1 active site
1.0   * between strands 3 and 5

E. Describe what an active site is, and why the position with the * is a possible active site.  (2.5 points)
Two criteria:
B. strand order reverses
1.0 COO- ends of 2 adjacent beta strands (i.e. loops) go in opposite directions.
0.5 active site = functional region   or  place to bind substrate.

The TATA box, a DNA sequence rich in Thymine and Adenine base pairs, is present in the basal promoter region of DNA.

2.0 Give a schematic drawing of the TBP (TATA-box binding protein). (4.0 points)
 

drawing should include: 1.0 saddle shape 1.0 5 antiparallel beta strands 1.0 2 helices 1.0 2 domains of above units

3.0 What section/part is involved in DNA binding?  Be specific. (3.0 points)

1.0 concave side
1.0 (central 8) beta strands
1.0 2 Phe pairs (partially insert between first two and last two bps of TATA - kink and partial unwinding - wide, shallow minor groove)

(C) What interaction binds TBP to TATA-box?  What was one proof that indicated this interaction? (3.0 points)

1.0 hydrophobic interactions
1.0 (15) side chains projecting from beta strands make hydrophobic contacts with the sugars and bases of DNA (interaction betw. underside TBP saddle and minor groove of DNA) 
   OR 
6 H-bonds betw 4 side chains from TBP and 4 H bond acceptors from bases in minor groove

(Note: this is different because most proteins interact with DNA in the major groove and are hydrophilic.)

1.0  Proof:  no water between TBP and DNA. 
 

As a first year graduate student your advisor, Dr. Dee N. Aye, asks you to isolate and characterize three different families of transcription factors from a mouse (eukaryotic) system.  Clearly describe to her the three kinds of transcription factor motifs you isolated, emphasizing how recognition and interaction are achieved by these proteins.  Drawing each motif might prove useful in explaining.

1. Helix-turn-helix: (3 pts)  Since you are in a mouse system- the student should describe homeodomain proteins (1.0 point). Describing or drawing the 2 alpha helices connected by short loops with helices 1 and 2 making up the ‘helix turn helix’ motif (1.0 point).  The second alpha helix (1.0 point) is the recognition site that interacts with the major groove of DNA 

2. Zinc Finger: (3 pts) Explaining or drawing a zinc finger (1.0 point), making sure to show / mention that the motif has a zinc atom, an alpha helix, and two beta strands (1.0 point). Must mention that it is the alpha helix that interacts with the major groove of DNA (1.0 point). 

3. Leucine Zipper:  (3 pts) Explaining or drawing two alpha helices (1.0 point) with the N-term region in a coiled-coil motif (1.0 point). Including that C-term alpha helices have basic or polar regions which interact at the major groove (1.0 point)

Must mention one of the following (1.0 point for one) general concepts about protein-DNA interactions somewhere in answer: Interactions with DNA are stabilized when the protein H-bonds with a PO4- on the DNA backbone. Side chains from the protein make salt bridges / ionic bonds with PO4- on the DNA backbone. Lastly, the protein has sequence specific recognition with DNA (forms H-bonds to the bases A T C or G). 

(A) Briefly describe the structure of the complex of human growth hormone 
bound to its receptor.  Your description should include recognized secondary 
structure motifs and the domain organization of each of the proteins 
in the complex.  In addition, draw a diagram of how the proteins fit together in 
the complex.  (4 points)

0.5 1 hormone + 2 receptor

Hormone:
0.5  4 helix bundle 
0.5  up-up-down-down topology
Receptor:
 0.5  two similar domains
 0.5  beta-sandwich motif
 0.5  one 3 strand, one 4 strand

1.0  Diagram  - showing hormone bound in a cleft in dimerized receptor.

(B) List the three protein-protein interactions in the complex, from 
strongest to weakest.  Given the relative strengths of these 
interactions, what sequence of events do the authors propose in their 
model of hGH receptor activation? (4 points)

1.0 Receptor 1 / Hormone strongest
1.0 then Receptor 2/ Hormone
1.0 Receptor 1/ Receptor 2 weakest

Sequence:  Hormone binds to R1, then R2. 

(C) Why were only the structures of the extracellular domains of the hGH receptor included, rather than the entire receptor? (2 points)

2.0 transmembrane proteins hard to crystallize. 

(A) Briefly explain two similarities and three differences between NMR spectroscopy and X-ray crystallography (the two techniques used to determine protein structure)? (4.5 points)

Similiarities include:  (MUST LIST THREE, 2.5 POINTS TOTAL, 1.0, 1.0, 0.5)
both require pure and concentrated protein, knowledge of amino acid sequence, computer, incorporation of markers (for x-ray:MIR, for NMR: isotopes), obtained through recombinant DNA technology.

Differences include:  (MUST LIST TWO, 2.0 TOTAL, 1 POINT EACH)
1.Form of protein to be examined: crystalline, few high quality crystals (x-ray) vs. protein in high conc. solution (nmr). 
2.  Size of protein:  protein can be virtually any size (x-ray) vs. protein is limited in sizeto under 30kD (nmr). 
3. Results:  electron density map used to build an atomic model of the protein (x-ray) vs.distance constraints between H atoms used to build a 3-D mol. model of the protein (nmr). 
4.  Type of info. obtained:  detailed structural info. of a "snapshot: of the protein (x-ray) vs. structural info. of a time resolved picture of the protein (nmr). 
5.  Requirements:  phase determination of the diffracted beams in which heavy metals are used, multiple isomorphous replacement (x-ray)vs. requires isotopes for unique identification of amide environment and distance (nmr).

(B) What type of information regarding protein structure is provided by: (3 pts)

a) COSY (1.5 pts if complete)
-gives peaks between H atoms covalently connected through one or two other atoms. Distance between H atoms should be less than or equal to three bonds. Give info. on interaction within the same amino acid: since the interactions differ for different types of amino acids, a fingerprint is obtained of each amino acid.  COSY allows the identification of the side chain.  Overall, reveals interactions between atoms that are covalently linked through one or two atoms on the same amino acid.

b) NOESY (1.5 pts if complete) 
-gives peaks between pairs of H atoms that are close together in space (less than or equal to 5 angstroms) even through they might be far apart in amino acid sequence.  Gives info. on interaction between H atoms from sequetially adjacent residues.  Gives distance constraints from specific H atoms in one residue to H atoms in another residue. Thus, NOESY reveals the 3-D structure of the protein

(C) Pick a letter from the list that is best described by the following statement: (2.5 points, 0.5 pt each)

a) NMR spectroscopy 
b) X-ray crystallography 
c) both 
d) neither 

I. Provides a "static" or "frozen" structural picture of the protein __B
II. Protein sample is prepared via the hanging drop method  _B
III. Requires amino acid sequence in order to better resolve the data obtained  C
IV - Requires isotopes such as 1H, 13C   __A
V. Exposes a protein to high intensity infrared rays __D

Zoink, a Martian immunologist is interested in the structure of IgG.  So he abducts you, (knowing that you are a learned student in structural biology) to teach him about the protein. 
Imagine that you are in the mother ship and answer Zoink's questions.

A. Describe the basic structure of IgG, the immunoglobulin fold (3.0 points if complete)

Student may draw and label, or describe.
Y-shaped OR T-shaped
Light and heavy chains
Constant and variable regions
Within variable there is the CDR regions, (three in each region)- loops that provide variability and generate antigen binding sites
3 + 4 antiparallel Beta sheets, held together by disulfide bonds, forms a barrel

B."Here is some of my previous work, BIBC 100 student. When I added papain, a proteolytic enzyme to the structure of IgG, it gave me three separate fragments.  Where would the enzyme cut to give me the three separate fragments?  What are these fragments?” (2 points)

Papain cuts at hinge region, yielding:

1.0  Two Fab fragments (light + heavy chain)
1.0  One Fc fragment (2 heavy chain)

C. What protein do T-cells recognize, and what are its functions? (2 points)

The T-cell receptors recognize MHC's. (1.0 point)
MHC function (1.0 points): present the partially digested antigen on infected cell surface, and thus signal to the T-cells destroy the infected cell.

C. Describe the basic structure of the class 1 MHC protein with bound peptide.

( 3.0 points, 1 pt for beta sheets as floor, 1pt for helices as sides, 1 pt for identifying where Ag binds)
The structure of MHC-I with bound peptide is (from top view), 2 domains of  4 anti-parallel beta-strands, and 1 alpha-helix for each (2 polypeptides).  Domains are associated such that the 2 antiparallel beta sheets form the “floor” and the helices cross over at the top and bottom to form the “sides”.  This creates a cavity for the antigen peptide.  See Figure 15.19.