Basic protein structure and stability II: Topics in side chain and backbone chemistry Basic anatomy

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Basic protein structure and stability IITopics
in side chain and backbone chemistry/Basic
anatomy of protein structure

• Biochem 565, Fall 2008
• 08/27/08
• Cordes

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Covalent chemistry of the side chains and the
backbone (main chain)

• reactions involved in enzymatic catalysis
• covalent posttranslational modifications that
  regulate activity and processing of proteins, and
  also cause covalent damage
• use of covalent side chain chemistry in analysis
  of proteins (e.g. crosslinking, attachment of
  labels, fluorophores etc)
• Use of side chain and backbone chemistry in in
  vitro peptide chemistry
• Both enzyme catalysis and posttranslational
  modifications will be covered later in the
  course. Some other important of the chemistry of
  side chains will also be covered in a class
  handout. In lecture today, we will cover just a
  few interesting, modern examples you may not
  hear about elsewhere.

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Chemistry of Cys thiol group has been exploited
in native chemical ligation (NCL) of peptides
The nucleophilic character of the thiolate anion
of Cys can be used to effectively catalyze
specific peptide bond formation between two
peptides, if a Cys is present at the N-terminal
end of one of the peptides, and if the other
peptide is labelled with a thioester at its
C-terminus. The Cys thiol exchanges with the
thioester, followed by S-N acyl transfer to the
amine group of the Cys, forming a peptide bond.
The limitation of this reaction is that it
requires Cys at the junction between the two
peptides you need an N-Cys peptide.
Tam et al Peptide Science 60, 194
(2001) Muralidahran Muir, Nature Reviews 3,
429 (2006)
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Protein splicing
translated sequence of a protein
N-extein
intein
C-extein
intein
C-extein
N-extein
splicing
intein
C-extein
N-extein
mature protein
excised intein
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Protein splicing mechanism
N-X acyl shift
transesterification
Asn cyclization
X-N acyl shift and succinimide hydrolysis
Perler Adam Curr. Opin, Biotech. 11, 377 (2000)
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Protein splicing
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Protein splicing
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Protein splicing
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Protein splicing mechanism at a glance
N-X acyl shift
transesterification
Asn cyclization
X-N acyl shift and succinimide hydrolysis
Perler Adam Curr. Opin, Biotech. 11, 377 (2000)
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Asparagine deamidation
Asparagine deamidation is a major route of
protein degradation and damage in vitro and in
vivo. It is of concern with regard to the purity
and proper function of peptide and protein
pharmaceuticals.
adapted from Xie L Schowen, RL J Pharm Sci 88,
8 (1999) See also Kossiakoff AA Science 240, 191
(1988)
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Oxidative damage/modification of methionine
Methionine oxidation is implicated in a variety
of aging-related disorders such as cataract
formation in the lens and Alzheimers disease,
though it may also serve nonpathological roles.
see for example Kantorow M et al. PNAS 101, 9654
(2004 Schoneich Arch Biochem Biophys 397, 302
(2002)
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The basic anatomy of protein structure
We will now discuss how we look
at/describe/analyze protein structures. For
Friday, I would like you to read the anatomy
section of the web version of JS Richardsons
classic 1981 article the Anatomy and Taxonomy of
Protein Structure, found at http//kinemage.bioc
hem.duke.edu/jsr/index.html This version
contains updated notes based on new structural
insights since the original article.
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The backbone conformation of proteinscombination
of regular and irregular structures
human hexokinase type I (1QHA) 102 kDa Rosano et
al. Structure 1999.
pig insulin (1ZNI) 5.7 kDa Bentley et al. Nature
1976.
These ribbon diagrams show the skeleton of a
protein. They are a smoothed representation of
the backbone or main chain structure and do
not show the side chains
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Backbone or main-chain conformation
There are three bonds between main chain atoms
(everything but the side chain) per residue, and
torsional rotation can occur about any of these
bonds, in principle. Hence, each residue has 3
angles that describe the main chain conformation
for that residue.
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Backbone conformationresonance forms of the
peptide group
The delocalization of the lone pair of the
nitrogen onto the carbonyl oxygen shown in the
resonance form on the right imparts significant
double bond character (40) to the peptide
bond. Breaking of this double bond character by
rotation of the peptide bond requires on the
order of 18-21 kcal/mol. Consequently there is
not free rotation around the peptide bond
rotation about the peptide bond happens on the
time scale of seconds/minutes--very slow
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Consequences of double bond character in the
peptide bond
the peptide C-N bond is 0.12A shorter than the
Calpha-N bond. and the CO is 0.02A longer than
that of aldehydes and ketones.
All six of the atoms highlighted at left lie in
the same plane, and as with carbon-carbon double
bonds there are two configurations--cis and trans
(trans shown at left)
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Consequences of double bond character in the
peptide bond
cis peptide bond
trans peptide bond
Still another consequence in the cis form, the R
groups in adjacent residues tend to clash. Hence
almost all peptide bonds in proteins are in the
trans configuration.
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...and that means that the dihedral angle
describing rotation around the peptide bond,
defined by the four atoms Ca(i)-C-N-Ca(i1), will
generally be close to 180. This angle is known
by the greek symbol w.
So the properties of the peptide bond place a
strong restriction on the backbone conformation
or main-chain conformation of proteins, that is
to say, the spatial configuration of the non
side-chain atoms.
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The peptidyl-proline bond
cis peptidyl-proline
trans peptidyl-proline
The peptidyl proline bond is an exception.
It can be in either the trans or cis
configuration, and the equilibrium constant
favors the trans only very slightly. Roughly
20 of all peptidyl proline bonds in native
proteins are in the cis configuration. This
is in part because the amide hydrogen is replaced
by a methylene group, which can clash with the R
group of the preceding amino acid. Remember
that there is still a kinetic effect on the rate
of isomerization of the peptidyl proline bond
that is similar to that for other peptide
bonds--proline cis-trans isomerization can take
seconds/minutes to occur, and this can actually
limit the rate at which a protein adopts its
native configuration beginning from a
disordered structure (more on this later).
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Backbone or main-chain conformation
So, one of the three degrees of freedom of the
protein backbone is essentially eliminated by the
properties of the peptide bond. What about the
other two?
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f and y angles
carboxy (C) terminal end
peptide planes
Torsional angles are defined by four atoms
y --gt Ni-Ca- Ci -Ni1
f --gt Ci-1-Ni-Ca- Ci
Notice that these are defined from N to C
terminus, using main chain atoms only. A
residues conformation is usually listed as
(f,y), since w is close to 180 for almost all
residues.
amino (N) terminal end
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• Unlike the peptide bond, free rotation occurs
  about the other two backbone bonds, but steric
  interactions within the polypeptide still
  severely limit plausible conformations, so that
  only certain combinations of phi and psi angles
  are allowed

and between carbonyls
or combinations of an R group, carbonyl or amide
group.
between R groups
180
not allowed
?
0
allowed
-180
?
-180 0 180
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Steric clashes disallow some f and y combinations
Theoretical calculations using hard sphere
approximations suggest which phi and psi
combinations cause clashes, and between which
atoms. Cross-hatched regions are allowed for
all residue types. The larger regions in the
four corners are allowed for glycine because it
lacks a side chain, so that no steric clashes
involving the beta carbon are possible.
from web version of JS Richardsons Anatomy
and Taxonomy of Protein Structure http//kinemage
.biochem.duke.edu/jsr/index.html
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Observed f and y combinationsin proteins
Phi-psi combinations actually observed in
proteins with known high-quality
structures. Gly residues are excluded from this
plot, as are Pro residues and residues which
precede Pro (more on this later). Contours
enclose 98 and 99.95 of the data
respectively. Notice that the observed
conformations do not exactly coincide with
the theoretically allowed/disallowed confs based
on steric clashes.
from Lovell SC et al. Proteins 50, 437 (2003)
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Sample Ramachandran plot for a protein
red allowed yellow additionally allowed pale
yellow generously allowed white disallowed
The squares denote non-glycine residues, while
the triangles are glycines. Glycines have no
side chain and are not as restricted because of
the lack of side chain steric clashes.
This protein has 219 residues, 90 of which are
in the allowed region and 10 of which are in
additionally allowed regions. None are in the
other regions (except glycines, which dont
count)