Transcript Document

Basic protein structure and
stability II:
Topics in side chain and backbone
chemistry/
Basic anatomy of protein structure
Biochem 565, Fall 2008
08/27/08
Cordes
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.
Chemistry of Cys thiol group has been exploited in native
chemical ligation (NCL) of peptides
HS
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 Nterminal end of one of the peptides,
and if the other peptide is labelled
with a thioester at its C-terminus.
SR
N
Tam et al Peptide Science 60, 194 (2001);
Muralidahran & Muir, Nature Reviews 3, 429 (2006)
CH
O
C
C
H2N
O
RSH
thiol-thioester exchange
S
CH2
O
N
CH
C
C
O
H2N
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.
CH2
S-N acyl transfer
HS
CH2
O
N
CH
C
C
N
H
O
N
C
= peptides to be joined together
translated sequence
of a protein
Protein splicing
N-extein
intein
C-extein
intein
N-extein
C-extein
splicing
N-extein
C-extein
mature protein
intein
excised intein
Protein splicing mechanism
HX
X = O or S
+H
H2N
H2N
+H N
3
3N
+H N
3
HN
X
HX
N-extein
N-extein
N-extein
O
intein
intein
O
O
intein
X
XH
XH
O
C-extein
O
C-extein
HN
HN
-O
-O
N-X
acyl shift
2C
H2N
O
C-extein
HN
-O
2C
2C
H2N
H2N
transesterification
O
O
O
H2N
+H N
3
H2N
HX
N-extein
+H N
3
O
HX
intein
X
C-extein
ligated
exteins
O
excised
intein
XH
H2N
-O
2C
O
HN
HO
-O
O
Asn cyclization
2C
hydrolysis
of succinimide
H2N
X-N acyl shift
and succinimide
hydrolysis
O
Perler & Adam Curr. Opin, Biotech. 11, 377 (2000)
Protein splicing
HX
X = O or S
+H
H2N
+H N
3
3N
HN
X
N-extein
N-extein
intein
O
O
intein
XH
XH
HN
HN
-O
O
C-extein
O
C-extein
-O
2C
2C
H2N
H2N
O
O
Protein splicing
H2N
H2N
+H N
3
+H N
3
HX
HX
N-extein
N-extein
O
O
intein
intein
X
X
C-extein
O
C-extein
O
H2N
HN
-O
-O
2C
2C
HN
H2N
O
O
hydrolysis
of succinimide
Protein splicing
H2N
+H N
3
HX
ligated
exteins
excised
intein
XH
O
HO
-O
2C
H2N
O
Protein splicing mechanism at a glance
H2N
HX
X = O or S
+H
+H N
3
H2N
+H N
3
3N
HX
HN
N-extein
X
O
intein
N-extein
N-extein
intein
X
O
O
intein
O
C-extein
O
C-extein
HN
-O
HN
HN
-O
O
C-extein
XH
XH
N-X
acyl shift
2C
H2N
-O
2C
H2N
2C
H2N
O
transesterification
O
O
H2N
H2N
+H N
3
+H N
3
HX
N-extein
O
HX
intein
X
C-extein
ligated
exteins
O
excised
intein
XH
O
H2N
-O
HO
2C
HN
-O
Asn cyclization
O
hydrolysis
of succinimide
2C
H2N
X-N acyl shift
and succinimide hydrolysis
O
Perler & Adam Curr. Opin, Biotech. 11, 377 (2000)
Asparagine deamidation
O
L-Asn peptide
C
O
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.
L-Asp peptide
NH2
H2C
N
H
O
C
O
O
H2C
NH
CH
C
N
H
H
N
CH
there is another
possible ringopened product
What is it?
C
O
O
hydrolysis
O
HO
NH3
C
O
C
O
H2C
H2C
N
N
N
H
CH
C
O
transient tetrahedral intermediate
adapted from
Xie L & Schowen, RL
J Pharm Sci 88, 8 (1999)
See also Kossiakoff AA Science 240, 191 (1988)
NH3
N
H
CH
C
O
succinimide intermediate
Oxidative damage/modification of methionine
O
O
HN
Methionine oxidation is
implicated in a variety of
aging-related
disorders such as
cataract formation in the
lens and Alzheimer’s
disease, though it may
also serve
nonpathological roles.
CH
C
HN
CH
C
O
HN
CH
CH2
CH2
CH2
CH2
CH2
CH2
S
CH3
oxidation
S
O
to sulfoxide
by various
reactive oxygen CH3
species
oxidation
to sulfone O
(generally
not reversible)
S
C
O
CH3
sulfone
reversible by methionine
sulfoxide reductases, thioredoxin
see for example Kantorow M et al. PNAS 101, 9654 (2004;
Schoneich Arch Biochem Biophys 397, 302 (2002)
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 Richardson’s classic 1981 article the
“Anatomy and Taxonomy of Protein Structure”, found at:
http://kinemage.biochem.duke.edu/~jsr/index.html
This version contains updated notes based on new structural
insights since the original article.
The backbone conformation of proteins:
combination 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
Backbone or main-chain conformation
O
H2N
CH
O
H
N
C
CH
R1
O
H
N
C
CH
R2

R3

residue 2
C


O
H
N
CH
C
O
H
R4


residue 3
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.
Backbone conformation:
resonance forms of the peptide group
O
..
C
R
R'
–O
N
C
H
R
+
N
R'
H
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
Consequences of double bond
character in the peptide bond
1.24 Å
H
O
123.2°
121.1°
R
121.9°
H2N
C
C
R
1.33 Å
H
C
N
OH
1.45 Å
H
O
H
O
R
H2N
C
C
R
C
OH
N
H
the peptide C-N bond is 0.12A
shorter than the Calpha-N bond.
and the C=O is 0.02A longer than
that of aldehydes and ketones.
H
O
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)
Consequences of double bond
character in the peptide bond
trans peptide bond
cis 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.
...and that means that the dihedral angle describing rotation around the
peptide bond, defined by the four atoms Ca(i)-C-N-Ca(i+1), will generally be
close to 180°. This angle is known by the greek symbol .
H
O
R

H2N
Ca
C
Ca
R
OH
N
H
residue i
H
O
residue i+1
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.
The peptidyl-proline bond
O
H
Ca
N
N
R H
cis peptidyl-proline
O
N
O
R H
H
Ca
N
trans peptidyl-proline
O
• 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 cistrans 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).
Backbone or main-chain conformation
O
H2N
CH
C
O
H
N
CH
R1
C
O
H
N
CH
R2
R3
residue 2
H
N
CH
C
R4


 ~ 180°
C
O

 ~ 180° 
residue 3
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?
O
H
 and  angles
carboxy (C) terminal end
Ri+1
peptide planes
Cai+1
H
Ni+1
Oi
C' i
i
i
Hi
Hi+1
H
Cai
Ci
Ni
C' i-1
Ri-1
Oi-1
Cai-1
H
amino (N) terminal end
Torsional angles are defined by
four atoms:
 --> Ni-Ca- C’i -Ni+1
 --> C’i-1-Ni-Ca- C’i
Notice that these are defined
from N to C terminus, using
main chain atoms only.
A residue’s conformation is usually
listed as (,), since  is close
to 180 for almost all residues.
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
allowed

not allowed
0
-180
-180
0

180
Steric clashes disallow some  and 
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 Richardson’s “Anatomy and
Taxonomy of Protein Structure”
http://kinemage.biochem.duke.edu/~jsr/index.html
Observed  and  combinations
in 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)
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 don’t count)