INTRODUCTION
• HDX is often used to probe protein structure and dynamics
using a variety of detection techniques (NMR, ESI MS, etc.)
• HDX is a complex process whose kinetics depends on both
protein dynamics and intrinsic amide hydrogen exchange rates
• Two distinct HDX regimes (EX1 and EX2) are commonly
identified depending on the rates of protein refolding (kcl) and
intrinsic exchange (kint)
• HDX under native conditions almost always follows the EX2
kinetics
• It is often assumed that denaturing conditions favor the EX1
regime (unless kint is reduced under such conditions even
more than kcl)
k1
k2
k-1
EX1: kint>>kcl: kHDX=kcl
k1
k2
k-1
EX2: kcl>>kint: kHDX=kintKunfold
• All of the above formalism has been initially developed to
interpret results of HDX NMR experiments and kcl refers to reprotection of individual hydrogen atoms, not collective motions
• When this formalism is applied to interpret the results of HDX
ESI (or MALDI) MS experiments, an implicit assumption is
made that kcl refers to a collective motion (e.g., transition from
an unprotected state to a structured conformation)
• Most proteins, however, are not simple two-state systems
and transitions within different pairs of states may result in
different HDX kinetics
• In order to interpret the results of HDX MS experiment
correctly, better understanding is needed on how complex
HDX kinetics reflects protein dynamics and structure
CASE STUDY 1: A TWO-STATE PROTEIN
(HDX kinetics of Chymotrypsin Inhibitor II)
• Chymotrypsin Inhibitor II (CI2) is a
small, single domain protein
• CI2 folds in a simple two-state
process under native conditions
• Non-native partially structured
states
of
CI2
(equilibrium
intermediates) have been detected
only under extreme conditions [1]
• Only two states (Native and
Unfolded) can be populated under
the conditions used in this work [2]
• The vast body of knowledge on CI2
dynamics
(NMR,
time-resolved
spectroscopy, etc.) makes it an
attractive model protein
HDX under native conditions: Two-Phase EX2 Kinetics
HDX was monitored across a wide range of solution
temperatures. It follows a simple biphasic kinetics up to 45oC.
Only one phase can be confidently measured at 60oC.
• The two phases correspond to ca. 15 fast exchanging amides
(often assigned to local fluctuations) and 30-41 slowly
exchanging amides (often assigned to global unfolding)
• The two rate constants can be used to calculate ∆G values of
these two processes
• Since ∆Gslow corresponds to a N → U transition, it can be used
to estimate free energy of protein folding
• Calculated ∆Gslow value is consistent with previous non-MS
data
∆Gslow (room T)
___________________________________________________
Technique used
fluorescence
(GdmCl denaturation)
fluorescence
(thermal denaturation)
HDX NMR (slow)
HDX MS (slow)
7.18±0.43
7.6
7.1
HDX NMR (fast)
HDX MS (fast)
4.95
4.6
7.03±0.16
___________________________________________________
Minimalistic energy surface diagram for a two-state protein
Monte-Carlo simulation of a particle movement on a 2-D energy surface
Infrequent events of
global unfolding (particle
leaves the potential well
for short periods of time)
Frequent events of local fluctuations
(particle climbs up the wall of the potential
well and reaches the “increased flexibility
region” for short periods of time)
• Elevation of free energy above the ground state by ∆Gfast
results in some increase of the chain flexibility, although the
protein remains in the potential well of the Native state
• Elevation of free energy above the ground state by ∆Gslow
results in a transition to an Unfolded state, providing
significantly more conformational freedom (random coil)
• Protein excursions from the bottom of the potential well are
very short, since the reverse activation energy barriers are
either small (U→N) transition or non-existent (local
fluctuations)
• Such behavior leads to EX2 type exchange kinetics, since
kcl>>kint for both processes
HDX under mildly denaturing conditions:
Mixed EX1/EX2 Kinetics
kHDX= kop= 0.04 min-1
• The EX2 component of HDX
kinetics appear to reflect rare and
short global unfolding events (very
large apparent ∆G; accurate
estimations are difficult due to
unavailable data on kint in the
presence of MeOH).
• The EX1 component of HDX
kinetics reflects “prolonged” global
unfolding events (proteins become
trapped in the U-state for extended
periods of time). The kinetic data can
be used to evaluate activation energy
of protein unfolding
Minimalistic energy surface diagram for a two-state protein
under mildly denaturing conditions
“Prolonged”
global
unfolding
events (particle leaves the potential
well and become trapped in the Ustate for long periods of time)
“Short” global unfolding events
(particle leaves the well for
short periods of time)
CASE STUDY 2: A THREE-STATE PROTEIN
(HDX kinetics of Ubiquitin)
• Ubiquitin (Ub) is a small, single
domain protein
• Ub folds in a simple two-state
process under native conditions
• A non-native partially structured
state of Ub (a molten globule-like Astate) can be populated across a
wide pH range at high alcohol
content [2]
• The vast body of knowledge on Ub
dynamics (including the A-state)
makes it an attractive model protein
HDX under native conditions: Two-phase EX2 kinetics
fast phase,
ΔGfast=3.1 kcal/mol
slow phase,
ΔGslow=5.4 kcal/mol
HDX follows a simple biphasic kinetics at room temperature.
Calculated free energy of protein folding (ΔGslow) agrees with
earlier denaturation measurements (ΔG=5.0 kcal/mol, [5])
Minimalistic energy surface diagram for a three-state protein
Monte-Carlo simulation of a particle movement on a 2-D energy surface
Frequent N→A transitions (low reverse
activation
energy
barrier
∆G‡A-N
prevents particle trapping in the A-state
for long periods of time)
Infrequent events of global
unfolding (particle leaves the
potential well for short periods of
time)
HDX of Ub under mildly denaturing conditions
• N → A: mixed EX1/EX2
• N, A → U: EX2
Minimalistic energy surface diagram for a three-state protein
under mildly denaturing conditions
“Short” global unfolding events
(particle stays outside of both Nand A-wells for short periods of
time) eventually returning to either
N- or A-basin of attraction
Both “prolonged” and short conformational switches
(long- and short-term excursions of the particle from
the potential well N to A-basin of attraction)
CONCLUSIONS
• Protein amide HDX reactions have very convoluted kinetics
which is reflective of multiple dynamic process
• HDX of small model proteins under native conditions reveals
two major types of dynamic events,fast local fluctuation and
slow global unfolding
• Free energies of both of these processes calculated based
on the kinetic data are consistent with that derived from other
sources
• Despite extensive efforts, no “pure” EX1 exchange
kinetics was ever observed even for small model proteins
• Mixed EX1/EX2 HDX kinetics of a two-state protein
reflects multiplicity of transitions between the native state
and the random coil
• Analysis of such kinetics yields valuable information on
the energetics of transitions between the native state and
the random coil
• Mixed EX1/EX2 HDX kinetics of a three-state protein
also reflects presence of multiple transitions between the
pairs of states (native state to non-native intermediate,
native state to random coil, etc.)
REFERENCES
1. Silow, M., Oliveberg, M. J. Mol. Biol. 2003, 326, 263.
2. Mohimen A., Hoerner, J., Dobo, A., Kaltashov, I.A. Anal.
Chem. 2003, in press.
3. Itzhaki, L.S.; Neira, J.L.; Fersht, A. R. J. Mol. Biol. 1997,
270, 89.
4. Jackson, S.E.; el Masry, N.; Fersht, A.R. Biochemistry
1993, 32, 11270.
5. Vijay-Kumar, S., Bugg, C.E., Cook, W.J. J. Mol. Biol.
1987, 194, 531.
ACKNOWLEDGEMENTS
NIH R01 GM61666
Dr. Andre Melcuk (UMass)
OVERVIEW
PURPOSE
Gain better understanding of how protein dynamics is reflected
by hydrogen exchange kinetics under various conditions.
METHOD
FT ICR MS
Amide Hydrogen Exchange (HDX)
RESULTS
• Protein amide HDX reactions have very convoluted kinetics
which is reflective of multiple dynamic process occurring on
various time scales within the protein
• Despite its extreme complexity, HDX can be used to map
protein energy surfaces and obtain quantitative information on
the energetics of protein structural dynamics