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 Open/ligand-free Adenylate kinase Closed: Ligand-bound

Conformational Change & Signaling Signaling protein: Calmodulin Calcium-free Calcium-bound Calmodulin, N-terminal lobe

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

Binding-curve perspective [oxygen] bound oxygen Fraction of

Physiological effects of cooperative oxygen-binding

Quantifying Allostery: Quasi-two-state model 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 Calcium-bound Calmodulin, N-terminal lobe

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