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Protein structure depends on amino acid sequence and interactions
Amino acid sequence
Local interactions
Long distance
interactions
Interactions between
subunits
Other methods?
Tools for protein topology studies
Complementary proteolysis
Identification of the amide bond cleaved by a single
proteolytic event that leads to complementary peptides
Selective chemical modification
Identification of the individual residue modified
by, even aspecific mono-bifunctional, reagents
Hydrogen/deuterium exchange
Quantitative analysis of amide bonds accessible
to solvent and available to exchange
Sequence/Structure
Paradigm
higher order structures
are
the
main
determinants of the
preferential
cleavage
site.
peptide bonds buried in
the protein core and/or
located
within
rigid
secondary
structures
are less accessible to
proteases
COMPLEMENTARY PROTEOLYSIS
Proteolytic
cleavage site
HPLC
ESMS
ARG 93
-lactalbumin
B
Abs
A
Proteolysis under
controlled conditions
C
C
A
A
Exposed and
flexible sites
C
B
min
Native protein
RP-HPLC
D
Abs
A
Proteolysis under
controlled conditions
Conformational
changes
A
D
min
Protein under different
experimental conditions
(ligands, denaturants, pH etc…)
LC- MS
LC- MS/MS
Proteolysis Experiment
Conditions
• Proteases
– Trypsin, Lys-C, Chymotrypsin, GluC, ………others
• Buffer condition
– pH, salt and detergent if necessary--Protein native condition?
• Protein concentration
• Time points
– 5min, 10min, 30min, 1h, 2hrs and 4hrs.
Complementary proteolysis
H/D exchange and mass
spectrometry
Very fast
R
O
R
O
N CH C N CH C
H
H
Very slow
Strongly depending on structural environment
Factors that Slow Exchange Rate
H/D-MS measures the
rate at which peptide
amide
hydrogens
exchange with the
hydrogen in the water
where the protein is
dissolved. The rate of
exchange reveals
the degree of exposure
of each amide
hydrogen in the folded
protein to water. In
the unfolded portions,
this rate is higher
than that of structured,
folded sections.
 Hydrogen bonding that creates the secondary structure, primarily
alpha-helices and beta sheets.
 Protection from the solvent, primarily due to being buried
in the hydrophobic core of the protein.
 Hydrogen bonding to water in the solvent (much smaller effect than
the previous two).
H/D exchange esperiments by ESIMS
D2O
Labelled
protein
Acid quenching
Protein
Before
ESI Probe
HPLC
After
Chung E.W. et al. (1997) Protein Science : 6, 1316 - 24
Labelled
protein
Pepsin digestion
5min, pH 2, 0°C
ESI Probe
HPLC
Labelled
peptides
Before
After
Wang L. et al. (2002) Mol. Cell.Proteomics: 1, 132-138
HAMLET
(Human Alpha-lactalbumin Made LEthal to Tumor cells)
APO
- Ca2+
HOLO
- Ca2+
Oleic acid
HAMLET
M. Svensson et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4221- 4226
Global H/D exchange profile
HAMLET
0%
1759.77
100%
1777,27
apo
0%
1759.77
100%
1777,27
Intensity (%)
15 sec
1 min
5 min
15 min
60 min
m/z
m/z
holo
0%
1759.77
100%
1777,27
Hydrogen -Deuterium Exchange
140
H/D exchanged
HAMLET
apo
holo
~130
~110
~80
apo*
20
0
60
Time (min)
Pepsin digestion for the local exchange profile
A
1
310
KQFTKCELSQ
LLKDIDGYGG
1
B
IALPELICTM
FHTSGYDTQA
IVENNESTEY
3
4
2
310
61
CKSSQVPQSR
NICDISCDKF
6
121 EKL
1
123
C
LDDDITDDIM
GLFQISNKLW
CAKKILDIKG
60
5
310
D
IDYWLAHKAL
CTEKLEQWLC
3
120
Holo α-lactalbumin
Apo α-actalbumin
HAMLET
100
80
Holo α-lactalbumin is always less
flexible than HAMLET and Apo
60
40
2
1
20
(12-23)
(1-11+118-123)
% exchanged protons
0
100
No substantial difference
between HAMLET and Apo in
peptides from the α domain
80
60
40
20
3
4
(24-31+103-107)
(41-52)
0
100
80
60
6
40
(60-96)
5
20
(53-59)
0
0
20
40
60
00
time (min)
20
20
40
40
60
60
HAMLET incorporates a higher
percentage of deuterium than
Apo in peptides from the β
domain
HAMLET/apo differential
topology
Arg70
Glu46
Glu49
Phe53
Tyr50
Asp37
Gln39
Chemical crosslinking
O
O
O
O
N O C CH2CH2CH2CH2CH2CH2C O
O
N
O
+
NH3
+
H3N
Saccharomyces cerevisiae Hsp26
 Hsp26 is an ATP independent cytosolic
chaperone of Saccharomyces cerevisiae.
Hsp26
expression
is
related
to
environmental stress (i.e. temperature).
Hsp26 acts as a buffer for (partly)
unfolded proteins, handing them over to
other chaperon-systems, such as Hsp70.
Hsp26 has the ability to form Dimers
and large Oligomers of 24 subunits.
Disassembling/Assembling of the oligomer plays a
vital role in chaperon function
ΔT
substrate
N
I
aggregate
ΔT
Hsp26
dissociated
Hsp26 oligomer
Hsp26-substrate complex
N
Chemical crosslinking
EDAC
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
Lys
O
CHCl-
NH2
CH3CH2
N
C
N
Glu, Asp
HO
(CH2)3
N+
CH3
EDAC
H
N
C
O
Legame isopeptidico
CH3
in situ digestion
MALDI-TOF analysis
Identification of crosslinked peptides
Chemical crosslinking
25°C
43°C
N-term. trimer domain
globular domain
α-crystalline domain
C-term. tail
116KDIDIEYHQNK126… 147VKVKESSSGK
156 158
151ESSSGKFK
23LLGEGGLRGYAPR35
190LKPQK194…198NHVKK20
2
N-term. trimer domain
globular domain
α-crystalline domain
C-term. tail