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Blueprint Series

Complete Solution
Chapter 3

3
PROTEIN STRUCTURE
AND FUNCTION

REVIEW THE CONCEPTS
1
...

The secondary structure of a protein is the various spatial arrangements that result from
folding localized regions of the polypeptide chain
...

Secondary structures, which include the alpha (a) helix and the beta (b) sheet, are held
together by hydrogen bonds
...
(The quaternary structure describes the
number and relative positions of the subunits in a multimeric protein
...
Despite the fact that folded proteins adopt conformations that are energetically
favorable, the amount of time required for a particular protein to arrive at this conformation on its own can vary significantly
...
Molecular chaperones func-tion to
protect an unfolded protein from participating in interactions that will take it off the
“pathway” to its native, functional conformation
...
However, chaperonins can also use encapsulation and ATPase
activity to give energetic “kicks” to misfolded proteins and get them back on the
pathway toward their native folded state
...
The active site of an enzyme is the region within which the substrate binds and is
converted into product
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The Michaelis constant

5

(Km) is equal to the substrate concentration at which an enzyme will generate
product at precisely one-half its potential maximal velocity
...
A rate constant never changes for a particular
enzyme
...
To calculate a rate (V), one would multiply the turnover
number (kcat) and [ES] (the concentration of enzyme-substrate)
...

4
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Therefore, the difference in free energy (DG) for a chemical does not
change as a consequence of adding enzyme
...
Therefore, E2 binds substrate with greater
affinity than E1
...
Although both E1 and E2 stabilize X‡ equally well, E2 binds more tightlyto S
than E1
...
In fact, the
reaction should proceed at the uncatalyzed rate in the presence of E2 because
the height of the barrier between the lowest energy state of the substrate and its
transition state is unchanged
...

5
...
As transition
states are, by definition, high-energy intermediates of chemical reactions rather
than stable molecules, one would first have to synthesize a stable molecule with
chemical properties similar to the transition state and use this “transition state
analog” as an antigen to promote an adaptive immune response in a test animal
...
Ubiquitin is a 76–amino acid protein that serves as a molecular tag for proteins
destined for degradation
...
This ubiquitination step is repeated many times, resulting in a long
chain of ubiquitin molecules
...
Proteasome inhibitors would be useful to treat cancers if they blocked
the degradation of proteins (e
...
, tumor suppressors) required to halt the progression of uncontrolled cell growth
...
Fribley and C
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Wang, Cancer Biol
...
, 2006 July 1; 5(7):745–8)
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Cooperativity, or allostery, refers to any change in the tertiary or quaternary
structure of a protein induced by the binding of a ligand that affects the binding

of subsequent ligand molecules
...
The activity of many proteins is regulated by
the reversible addition/removal of phosphate groups to specific serine, threonine, and tyrosine residues
...
Phosphorylation/dephosphorylation
changes the charge on a protein, which typically leads to a conformational change
and a resulting increase or decrease in activity
...

8
...
In this separation technique, known
as rate-zonal centrifugation, proteins of larger mass generally migrate faster than
proteins of smaller mass
...
Gel electrophoresis can also separate proteins based on their mass
...
Because the
migration of proteins through a polyacrylamide gel is also influenced by shape of
proteins, the ionic detergent sodium dodecyl sulfate is added to denature proteins and force proteins into similar conformations
...

9
...

In gel filtration chromatography, the protein solution flows around the spherical
beads and interacts with depressions that cover the surface of the beads
...
In
ion-exchange chromatography, proteins are separated on the basisof their charge
...
Positively charged proteins will bind to
negatively charged beads, and negatively charged proteins will bind to positively
charged beads
...
The protein
solution is passed over the beads and only those proteins that bind to the ligand
attached to the beads will be retained, while other proteins arewashed out
...

10
...
Methionine or cysteine labeled with sulfur-35 are two commonly used radioactive amino acids, although many others
have also been used
...
In one example of this technique, cells are labeled with a radioactive compound and then overlaid with a photographic emulsion sensitive to

radiation
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A Western blot is a method for
detecting proteins that combines the resolving power of gel electrophoresis, the
specificity of antibodies, and the sensitivity of enzyme assays
...
The proteins are
then transferred onto a nylon filter
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The presence of this protein-primary antibody-enzyme-conjugated
secondary antibody complex is detected using an assay specific for the conjugated enzyme
...
X-ray crystallography can be used to determine the three-dimensional structure
of proteins
...
The
diffraction pattern generated when atoms in the protein scatter the x-rays is a
characteristic pattern that can be interpreted into defined structures
...
A low dose of electrons is used to generate
a scatter pattern that can be used to reconstruct the protein’s structure
...
From the magnitude of the effect of one atom on
an adjacent atom, the distances between residues can be calculated to generate a
three-dimensional structure
...
The principal
disadvantage of x-ray crystallography is the challenge of producing samples in
the form of single crystals suitable for diffraction experiments
...
It also
is ideal for monitoring protein dynamics
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The principal advantage of electron microscopy is the relative ease of sample preparation
...
NMR is better for small proteins
...

12
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Basically, the
investigator would collect protein samples from the cancerous cells and from
the normal healthy cells, the latter serving as a control
...
If a protein “spot” were present in the sample from the
cancer cell and not the control, it would be isolated out of the gel, proteasedigested using trypsin to generate peptides that are mixed with a matrix, and
applied to a metal target
...
In the case of a time of flight (TOF) mass
analyzer, the time it takes the ions to pass through the analyzer before reaching

the detector is inversely proportional to its mass and directly proportional
to the charge they carry, generating a spectrum in which each molecule has
a distinct signal, allowing the investigator to calculate each ion’s mass
...
The
mass and charge signature, or fingerprint, of the unknown is compared to
that of peptides in a database and the best match protein is identified



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