HDX and protein dynamics - University of Massachusetts Amherst

20 Pages·2003·0.95 MB·English

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 (k ) and
cl
intrinsic exchange (k )
int
• HDX under native conditions almost always follows the EX2
kinetics
• It is often assumed that denaturing conditions favor the EX1
regime (unless k is reduced under such conditions even
int
more than k )
cl
k k
k
1 2
-1
EX1: k >>k : kHDX=k
int cl cl
k k
k
1 2
-1
EX2: k >>k : kHDX=k K
cl int int unfold
• All of the above formalism has been initially developed to
interpret results of HDX NMR experiments and k refers to re-
cl
protection 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 k refers to a collective motion (e.g., transition from
cl
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 G corresponds to a N U transition, it can be used
slow
to estimate free energy of protein folding
∆
• Calculated G value is consistent with previous non-MS
slow
data
∆
Technique used G (room T)
slow
___________________________________________________
fluorescence

(GdmCl denaturation) 7.03±0.16
fluorescence
(thermal denaturation) 7.18±0.43
HDX NMR (slow) 7.6
HDX MS (slow) 7.1
___________________________________________________
HDX NMR (fast) 4.95
HDX MS (fast) 4.6
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 Frequent events of local fluctuations
global unfolding (particle (particle climbs up the wall of the potential
leaves the potential well well and reaches the “increased flexibility
for short periods of time) region” for short periods of time)
∆
• Elevation of free energy above the ground state by G
fast
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 G
slow
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
k >>k for both processes
cl int
HDX under mildly denaturing conditions:
Mixed EX1/EX2 Kinetics
kHDX= k = 0.04 min-1
op
• The EX2 component of HDX • The EX1 component of HDX
kinetics appear to reflect rare and kinetics reflects “prolonged” global
short global unfolding events (very unfolding events (proteins become
∆
large apparent G; accurate trapped in the U-state for extended
estimations are difficult due to periods of time). The kinetic data can
unavailable data on k in the be used to evaluate activation energy
int
presence of MeOH). of protein unfolding