Lecture 9. Protein stability and folding
Thursday 14 February 2018
Protein stability. Factors that contribute to protein stability. The Levinthal paradox implies that proteins fold by directed pathways. General principles of protein folding. Protein folding in vivo: Molecular chaperones. Diseases due to protein misfolding.
Reading: Lehninger - Ch.4, pp.142-151.
Summary
Reading summary. Proteostasis. Loss of protein structure results in loss of function. Protein denaturation. Amino acids sequence determines tertiary structure. Anfinsen experiment. Polypeptides fold rapidly by a stepwise process. The "folding funnel" diagrams. Some proteins undergo assisted folding. Chaperones: Hsp70, chaperonins (GroEL/ES), PDI, PPI. Defects in protein folding provide the molecular basis for a wide range of human genetic disorders. Box 4-6 -Medicine- Prion diseases.
Factors that contribute to protein stability - a summary
By protein stability we mean that the free energy of the process
unfolded protien → folded native protein
has a negative value. The factors that stabilize the native state of naturally-occurring proteins can be summarized as follows:
- Residues with stereochemically favorable conformations
- Polar groups (e.g. main-chain amide and acyl oxygens) are paired in H-bonds
- Burial of hydrophobic surface area within the interior along with efficient, dense packing to maximize favorable van der Waals contacts
Let's consider each of these factors in turn, and in doing so we will see their relationships to our previous summary of "invariant" features of protein structure. As Lesk points out, "(t)he fact that one conformation of the protein - the native state - has subtantially greater stability than others is complex but not mysterious."
For main chain conformations, the first factor is reflected in the energy landscape of the Ramachandran plot. The conformations of side chains tend to cluster in recurring patterns called rotamers. The dihedral angles displayed in standard side chain rotamers are generally those corresponding to potential energy minima in a conformational analysis. For example, the side chain dihedral angles χ1, χ2,.. (in the general notation for side chain dihedral angles, χ1 is the dihedral angle for the bond between Cα and Cβ, χ2 is the dihedral angle for the bond between Cβ and Cγ, etc.) for an aliphatic side chain tend to have values corresponding to gauche and anti conformations as would be expected from an analysis of the C2-C3 bond of butane (see, e.g., Solomons & Fryhle Organic Chemistry 7th ed. p.153). The general guiding principle that underlies this first factor is that steric collisions raise the energy of a structure and lower its stability, and is encompassed within the "invariant" structural feature of the overall lack of strain in stable native protein structures.
The second factor arises from the thermodynamic instability of charged and polar groups in nonpolar environments, relative to an aqueous (polar solvent) environment. If the polar groups of the main chain are not in paired interactions, the protein will prefer the denatured state in order to allow these groups to hydrogen bond with solvent. Note that the pairing of polar main chain groups is neatly accomplished within the regular secondary structure patterns of α-helices and β-sheets.
The third factor is closely related to both the hydrophobic effect and the maximization of van der Waals interactions in protein interiors. It is possible to gain the entropic benefit corresponding to the hydrophobic effect by simply excluding water from the protein interior. This can be accomplished by a structure possessing no channels wider than the "radius" of a water molecule (taken to be about 1.4 Å). But this does not necessarily maximize the enthalpic benefit of close-packing of side chains within the core.
The Levinthal paradox
It is plausible that evolution has acted to select for amino acid sequences that fold into a well-defined native state that (despite the overall modest stability of the folded state) is well-separated in free energy from other conformations. The Levinthal paradox suggests that sequences may have also been selected for on the basis of well-defined folding pathways. The essential idea behind it is that if proteins had to fold via a simple, random conformational search, the time required - calculated using reasonable assumptions for the number of available conformations and the speed of bond rotations required to change conformations - is astronomically vast. The obvious implication is that polypeptide chains do not try out all, or even a significant fraction, of the possible conformations, and that protein folding is somehow a directed process.
Protein folding
In contrast to our reasonably complete understanding of protein stability, the actual process of protein folding remains somewhat mysterious. The relatively small values of ΔG for the folding transition and the heterogeneous nature of the dentured state - not to mention the rapidity by which the process occurs - make it difficult to elucidate distinct folding pathways.
General principles of protein folding
1. Many proteins appear to fold in distinct stages. The initial stage is a quite rapid "burst phase", occurring on a millisecond timescale, and involves a dramatic reduction in the volume occupied by the polypeptide chain, representing its conversion from a random coil to a so-called molten globule state. This stage is described as a hydrophobic collapse since the molten globule state is characterized by a segregation of nonpolar groups to exclude water and form a nascent hydrophobic core. The molten globule differs from the final native state, however, in its less compact and more dynamic nature. The following slower stage(s), which typically occur on a timescale of a few seconds, establish a more compact core and further stabilize secondary structural elements, while specific docking between supersecondary structures occurs. Formation of disulfide bonds and isomerization of X-Pro peptide bonds occur at a late stage for some proteins.
2. A "folding funnel" energy landscape. Progression of protein folding by conformational adjustments follows a path down an energy landscape in which the energy of the polypeptide chain decreases, as well as its entropy, as the number of conformations available to the chain becomes more limited. The energy landscape is modeled by a "folding funnel", in which height represents energy of a conformation, while the width of the funnel at a given height represents the number of conformations with that energy, i.e. the entropy of the polypeptide.
3. Protein folding is hierarchical. The hierarchical view of native protein structure suggests that some aspects of these hierarchies apply to folding. Indeed, a number of lines of evidence support the idea the folding process occurs in a hierarchical fashion. In this view, local interactions determine secondary structures, and in turn secondary structure elements seem to be determined by adjacent blocks of primary structure and their context-dependent secondary structural tendencies.
4. Folding in vivo. Proteins are synthesized by the ribosome in an N to C direction. This suggests that a sequence of local interactions are the initial events in folding, and that the results of these local interactions within the nascent N terminus provide the context for succeeding local interactions in the early "burst phase" of folding. Furthermore, some proteins - particularly large, multidomain proteins with complex structures - fold slowly or inefficiently. Protein folding in vivo is assisted by a variety of folding accessory proteins. The molecular chaperones are essential proteins that bind to nascent and misfolded polypeptides and promote proper folding, while protein disufide isomerase and peptidylprolyl isomerase help proteins escape from configurations with the wrong disulfide pairings or X-Pro peptide isomers. In fact, efficient folding in vitro is only observed in favorable cases - usually with small, single domain proteins with high stability like RNase A. More typically, denatured proteins fold slowly and with low efficiency.