2007/9/11 4:53:00 PM
The Second Qualification Exam (2006) for NTHU
PART 1
1. The structural motifs formed by the association of two or more helices are
categorized as supersecondary structures. Draw the following
supersecondary structures in proteins. (1). -, (2). --, (3)  hairpin, and
(4) Greek key. (8%)
2. Name four of the bonds between two atoms from the strongest to the
weakest. (4%)
3. Define the following terms (1) protein domain, (2) protein fold, and (3)
protein quaternary structure. (3%)
PART 2
1. If you intend to express a foreign protein for the assay of its function, what is
your concern?
For example, what are the expression systems that will be used and what is
your reasoning; do you have to consider posttranslational modifications? If
so, what should you do, and so on so forth?
PART 3
1. Use proton as an example, please describe the occurrence of NMR briefly.
(6%)
2. Please define briefly the following terms.
(a) Spin echo
(b) Spin-lattice relaxation time
(c) Nuclear Overhauser enhancement
(d) Free induction decay
(e) Pulse sequence
(f) Solvent suppression
(g) Magic angle spinning
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(2% each)
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PART 4
1. A constructive interference (reflection) is seen only when Bragg’s equation
(or Bragg’s law) is satisfied for a particular set of Bragg planes. Please use
figure and equation to explain the Bragg’s law. (5 point)
2. For Fourier synthesis to work, we need to know the phases for all reflections.
For a long time, obtaining phases was one of the rate limiting steps in
crystal structure determination. To date, there are several methods to solve
the phase problem. Please list three major methods in the phase
determination of the protein crystallography and explain the principle,
requirement and the limitation in the each method. (10 pts)
.3. (a) What is anomalous scattering? (b) Please use a diagram and equation
to explain why anomalous scattering can breakdown the Fridel’s law. (5
point)
PART 5
1. 2D gel electrophoresis (2-DE) is the most commonly used separation
technique today, (1) please describe the separation principle (2) Can you
use the following example to draw a representative 2DE map for
comparative experiment.
Example, you want to look for proteins that are differentially expressed in
Hela cell after stimulation of growth factor (10 points)
2. For identification of protein, mass spectrometry has become one of the
core technologies for unknown protein identification. Please describe one
of the following principles: (a) Peptide Mass Fingerprinting or (b) Tandem
mass spectrometry. (5 points)
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PART 6
1.
As we know that the native tertiary structure of proteins/enzymes is
stabilized by number factors. Please
(a) provide the names and chemical structures of the four amino acid
side-chains involved in “salt bridges”. (4 points)
(b) elucidate the identity of the singular covalent “cross link” that is
occasionally found. Why do you think Mother Nature had to resort to the use of
such linkages? (2 points)
2.
Match each statement below with the most relevant concept by placing
the appropriate letter in the boxes. (6 points)
A Induced-fit theory
B kcat
C Zipper principle
(a) Represents the maximum number of substrate molecules converted
to product molecules per active site per unit of time, that is, the
number of times the enzyme “turns over” the substrate to the product
per unit of time.
(b) Binding of substrate triggers a change in enzyme conformation from a
catalytically inactive to a catalytically active one.
(c) Stepwise binding of flexible, high molecular weight ligands to their
receptors.
3.
Consider a particular zinc carboxydipeptidase (cleaves dipeptides from
the C-terminus of proteins), which specifically recognizes a tyrosine
residue at the penultimate position. Some measured kinetics parameters
for the “specific substrate” N-(acetyl)glycyl-L-tyrosyl-L-valine (1) and some
relatively poor substrate analogues, as well as for some inhibitors are
listed below.
Km (mM)
3
Vmax (sec-1)
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Ac-Gly-L-Tyr-L-Val-OH (1)
0.70
Ac-Gly-L-Ala-L-Val-OH (2)
Ac-Gly-L-Tyr-L-Val-NH2 (3)
0.40
5.0
3500
120
2100
Ki (mM)
Ac-NHCH2SO2-L-Tyr-L-Val-OH (4)
Ac-NHCH2SO2-L-Tyr-L-Val-NH2 (5)
0.03
0.41
no turnover
no turnover
(a) What factors do you think rationalize why 2 and 3 are poor substrates
relative to 1? (Recall that “goodness” of a substrate is represented by V max/Km
and other specific interactions as well as IBE) (3 points)
(b) Why are 4 and 5 good inhibitors? (2 points)
(c) Based on the molecules shown above, you might go about raising
monoclonal antibodies which catalyze the same reaction as above with the
exception that dipeptides are cleaved specifically when lysine rather than
tyrosine is present at the penultimate position. Please provide the appropriate
structure of the ideal competitive inhibitor of your antibody catalyst. (2 points)
(d) In the Michaelis-Menton plot below, the uninhibited reaction is described by
curve 1. The effect of a competitive inhibitor is represented by curve: (1
points)
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