Lecture 11: Protein Folding & Stability Margaret A. Daugherty Fall 2003 Structure - Function Protein Folding: What we know 1). Amino acid sequence dictates structure. 2). The native structure represents the lowest energy state for a protein (physiological conditions). 3). Proteins are densely packed as small organic crystals. Protein Folding 4). A protein cannot sample all possible conformations in finding its native structure (Levinthal s paradox). 5). Protein folding in vitro is a good model for in vivo folding. 6). Protein folding is a cooperative process, usually between N <--> U states. 7). Intermediates with non-native structure can exist is some protein folding pathways. 8). The molten globule is likely to be an intermediate on protein folding pathways. The Future of Folding 9). The protein folding problem will be solved within 5 years (Walter Gilbert, 1988) 10). Designed proteins usually turn out to be molten globules. 11). We will eventually be able to predict protein structure from sequence.
Anfinsen s protein folding experiment Ribonuclease A 124 aa, pancreatic enzyme 1916-1995 Anfinsen s experiment: sequence dictates structure `57: Nobel Prize in 1972 (1) a. Reduce protein (β-me destroys disulfides) b, Unfold protein in urea (2) Remove urea - allow protein to refold Remove β-me- allow disulfide to reoxidize (1) 100 % activity (2) (4) ~1% activity (3) Remove β-me - allow disulfides to reoxidize Remove urea - alllow protein to refold (4) Add trace β-me, warm ~10 hrs ==> 100% ACTIVITY Add trace β-me + cytosolic* fraction ~2 minutes ==>100% ACTIVITY 0% activity (3)
PROTEIN FOLDING IS ENERGETICALLY FAVORABLE Unfolded Nucleation of secondary structural elements Interaction of secondary structural elements molten globule? Native Energetic contributions to protein folding Conformational entropy Hydrogen bonds Electrostatic Forces *The hydrophobic effect* van der Waals Forces Other forces
CONFORMATIONAL ENTROPY Boltzmann s Equation: S = k ln W VS. The polypeptide chain in the unfolded state has more conformational freedom than the folded state. HYDROPHOBIC EFFECT Hydrophobic effect: Thermodynamic consequence due to avoidance of H 2 0 by the apolar side chains of a protein. Predicted in 1959 by Kauzmann (before 3D structures). Major contributor to stabilization of native state: proportional to apolar surface area buried.
Entropy of protein folding has unfavorable and favorable contributions! ordered H 2 0 about hydrophobes result in clathrate structures Sequestering of hydrophobes to interior of protein molecule => Release of H 2 0 HYDROGEN BONDS IN PROTEINS Almost all groups capable of hydrogen bonding are, in fact, hydrogen bonded Baker & Hubbard, 1984
H-bond contribution to native state free energy may be small. Unfolded Folded H + H-O-H 0-H Stabilizing force only if protein-protein and water-water H-bonds are more favorable than those of protein-water ELECTROSTATIC FORCES Charge-dipole: U qm = qmcosθ/εr 2 m = dipole moment ε = dielectric constant Charge-charge ion pairs Charge-dipole amide-helix dipole helix-helix ion-helix
van der Waals Forces non-polar interactions or dispersion forces: due to local fluctuations in electron density U = A/r 12 - B/r 6 r Repulsive potential due to bringing two atoms so close that their electron clouds interpenetrate Attractive potential due to mutual induction of electrostatic dipoles. These attractive forces are weak. However, they are additive and can make a significant contribution to stability when summed over a molecule Folding Also Involves Arrangement of S-S bonds BPTI: bovine pancreatic trypsin inhibitor
Contributions to the native state free energy For a typical small globular proteins, the favorable and unfavorable interactions are energetically enormous (~100s kcal/mol) but the difference in free energy from unfolded to native state is small. KINETIC COMPONENT: LEVINTHAL S PARADOX AND TIME Given an polypeptide sequence, how does it fold into a native conformation in a reasonable amount of time? Example of ribonuclease (124 amino acid residues): if we assume each residue can sample 3 conformations ~10 50 conformations If folding samples a new conformation every 10-13 seconds 10 30 years to sample all conformations In vitro: ribonuclease folds in ~1-2 minutes
The native structure of a protein represents its lowest energy state The energy landscape of protein folding can be visualized as a funnel in which many paths to the lowest energy state, e.g., the folded state are possible. The paths through the funnel represent kinetic pathways. A. Rapid folding pathway B. Protein goes through an energy minimum, which may slow folding. unfolded folded ENERGY DECREASE More realistic model IS PROTEIN FOLDING IN VITRO A GOOD MODEL FOR PROTEIN FOLDING IN VIVO? PARADOXES OF PROTEIN FOLDING: RATES OF CORRECT FOLDING: in vivo: few minutes in vitro: several hours EFFICIENCY OF CORRECT FOLDING: in vivo: ~ 99% in vitro: a few % for many OFTEN, in vitro REFOLDING OF POLYPEPTIDE CHAIN DOES NOT RESULT IN NATIVE STRUCTURE
ACCESSORY PROTEINS PLAY AN IMPORTANT ROLE IN NASCENT PROTEIN FOLDING Conceptual transition from self-assembly to assisted assembly principle of protein folding Foldase: catalyze the necessary covalent reactions directly involved in protein folding. Chaperone: assist in the correct non-covalent folding & assembly of nascent peptides. Both classes accessory proteins function by manipulating the target conformation and are catalytic. PPI: peptidyl prolyl cis-trans isomerase Steric and resonance effects favor trans configuration 10-20% of prolines exist in cis configuration Problem: prolines isomerize freely in Unfolded state Bigger problem: rate constant for isomerization is slower than folding Biggest problem: prolines in wrong configuration cause misfolded proteins
CHAPERONINS: GroEL-GroES complex GroEL: 2 ring structure of 7 subunits GroES: ring structure with 7 subunits GroEL-GroES: STRUCTURAL CONSIDERATIONS GroES interaction alters conformation of the GroEL subunit, resulting in a correspondingly larger binding cavity
GroEL 1). Non-native protein binds to the trans ring of GroEL-GroES complex. GroEL-GroES MECHANISM GroES 2). End-to-end exchange of GroES (through symmetric intermediate?). Substrate encapsulated in trans cavity. 3). In presence of ATP- productive folding of substrate protein occurs. trans 4). Release of GroES and native protein. GroEL-GroES complex is regenerated for next cycle. cis Denaturation Loss of 3D structure; Heat affects weak interactions (H-bonds); sharp transition suggests cooperativity; ph alters ionization states of AAs; Detergents interfere with hydrophobic interactions; Chaotropic agents (urea, guanidinium chloride) increase solubility of non-polar side chains in H 2 O.
What is Levinthal s paradox? What does a folding funnel describe? Review What is the sequence of protein folding? What is a molten globule? What are the enthalpic and entropic contributions to protein folding? Is protein folding in vitro a good model for understanding in vivo folding? What are the two classes of accessory proteins involved in protein folding? How does GroEL-GroES mediate protein folding?