Conformational Change in Proteins
Molecular Biophysics III
Prof. Daniel M. Zuckerman,
Dept. of Computational Biology
Conformational Change & Function
• Many (most?) proteins function via
conformational changes
• Outline
– Ensemble Picture and Examples
– Hemogolobin and allostery
– Myosin, kinesin and motion
– Functional motif for NTP hydrolysis
– Calmodulin and signalling
• Reference/reading: Berg et al., Biochemistry
– Also source for figures
Ensemble Picture (Statistical Mechanics)
• An ensemble of paths, traversing ensembles of
intermediate structures, connects two
ensembles of structures
intermediates
structural ensemble A
structural
ensemble B
Motor proteins
Myosin
Fluctuations in Biology
• Regulation: fatty acid binding proteins
Enzyme Conformational
Change
• Adenylate kinase
Open/ligand-free
Closed:
Ligand-bound
Conformational Change & Signaling
• Signaling protein: Calmodulin
Calcium-free
Calmodulin,
N-terminal
lobe
Calcium-bound
More dramatic conformational flexibility
• Open and closed Ca-bound calmodulin
– Likely both occur in solution … and everything “in
between”
Calcium-bound
Calcium-bound
Consequences of Induced Fit Idea
• The idea: Ligand binding induces
conformational change
• Some possibilities:
– Ligand binds to an apo-like or holo-like
configuration
– Ligand unbinds from holo-like or apo-like
configuration
• One way or another, proteins must undergo
large conformational fluctuations
– And this must happen all the time to allow constant
binding and un-binding
Allostery: “cooperativity” in binding
• For proteins with more than one binding site,
the binding events often are not independent
– Even when binding sites are identical!
– Conformation & affinity change as additional
ligands bind
– This is allostery
– Hemoglobin is the classic allosteric protein
Note: some of
these states may
not exist
(stably).
Hemoglobin structure
• Four sub-unit homodimer (a,b)2
FYI:
Chien Ho
at CMU
Hemoglobin: heme structure
• Oxygen transported via integral heme groups
– Four hemes, four binding sites
– This small change triggers macroscopic motion
Fraction of
bound oxygen
Binding-curve perspective
[oxygen]
Physiological effects of
cooperative oxygen-binding
Quantifying Allostery: Quasi-two-state model
T
R
• MWC model (Monod, Wyman, Changeux)
• Equilibrium between T, R -- each of which have four
(static) identical binding sites
–
–
–
–
R = relaxed conf, T = tense conf., S = substrate
Conf. equil: R  T, with eq. const. L = [T]/[R]
Bind. equil. 1: R + S  RS1, with KR/4 = [R][S]/[RS1]
Bind. equil. 2: T + S  TS1, with KT/4 = [T][S]/[TS1]
• Factor of 4 since 4 sites to bind
– Further equilibria: TS1 + S  TS2
MWC Model can be “solved”
• Solve with paper and pencil (no computer!)
– Yields prediction for fraction of bound sites as a
function of oxygen concentration
• Highly successful for hemoglobin
• Inadequate for some systems:
– Omits sequence-dependence
– Alternative model: KNF
• Fersht, Ch. 10
Motor Proteins: Myosin
(kinesin)
Myosin structure
(ATP analog)
Binds to actin
Myosin: the structural trigger
• Again, a tiny
change triggers
large-scale motion
Myosin-Actin Interactions
• Figs from Alberts, Molecular Biology of the Cell
Kinesin structures
• Kinesin expert at Pitt: Susan Gilbert (Biological
Sciences)
Kinesin trigger
P-loop structural motif
• For hydrolyzing NTP (to NDP)
– N = nucleotide
Re-connect with statistical mechanics
• Timescales and barriers
– Rate as attempt frequency and Arrhenius factor
• Multiplicity of pathways
– Partial basis for ensemble picture (in addition to
dynamic variability)
intermediates
structural ensemble A
structural
ensemble B
Structural Analysis of Calcium Signalling
• Calmodulin is unusual
– Ca2+-bound state is “open” -- solvent exposed
• Hydrophobic residues exposed to solvent!
– Contrast to enzymes which often “close” to envelop
substrate
Calcium-free
Calmodulin,
N-terminal
lobe
Calcium-bound
Why CaM exposes hydrophobes
• Hydrophobic surface bind other proteins to
continue signalling cascade
Note: two conformational changes -- second is open-to-closed!
“Generalized Allostery”
• Nussinov & coworkers in recent Proteins
• Nearly all proteins can be considered allosteric
– So long as interactions shift equilibrium
• Calmodulin easily fits into this view
– Calcium switches conformational equilibrium to
open state
– Open stat favors peptide binding