Lecture 14 Summary

Text highlights: The somewhat marginal thermodynamic stability of most proteins is mainly accounted for by the hydrophobic effect, with significant contributions from electrostatic interactions. Hydropathy plots; ion pairs. Disulfide bonds help stabilize backbone conformations, but they occur almost exclusively in extracellular proteins. Small protein domains can be stabilized by metal ions, as is seen with the zinc finger motif. Proteins are dynamic: their tertiary structures flex, or "breathe", and in some cases undergo large-scale conformational changes (6: 156-158).

Protein denaturation and renaturation. Thermostable proteins (box). Anfinsen's experiment on RNase A and the role of correct disulfide bond formation (6: 159-160).

Protein folding: Levinthal's paradox; Proteins follow folding pathways. The energy-entropy diagram, or "folding funnel". Protein structure prediction and protein design (box). The role of protein disufide isomerase (PDI) (6: 161-165).

Molecular chaperones assist protein folding in vivo. The GroEL/ES chaperonin structure and function (6: 165-168).

Protein misfolding as a cause of disease. Amyloid-β protein accumulation in Alzheimer's disease. Infectious prion diseases. Amyloid fibrils are β-sheet structures (6: 168-172).

Factors that contribute to protein stability - a summary

The factors that stabilize the native state of naturally-occurring proteins can be summarized as follows:

• All residues must have stereochemically allowed conformations
• Buried polar atoms must be paired in H-bonds
• Enough hydrophobic surface area must be buried and the interior must be sufficiently densely packed to provide thermodynamic stability

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.

Sequence determines structure

Refer to the discussion of Anfinsen's experiment on ribonuclease (VVP3e, pp.159-160) which captures the essential ideas. Further details from lecture will be added when time permits. This is an important concept, so be sure you understand it and why the example of the mature form of insulin is an apparent - but not genuine - exception to this rule.

The Levinthal paradox

I have made the argument in class 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.

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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.

Learning objectives

• Recommended: Learn how to use a structure viewing program (such as PyMOL or Swiss PDB Viewer).
• Describe and explain general features of protein tertiary structure.
• Explain the meaning of the phrase "sequence determines structure".
• State the Levinthal paradox, and explain its implications for protein folding.
• Describe the general principles of protein folding.
• Recognize the relevance of protein folding and stability to a class of disease-causing proteinopathies

Page updated 10-05-10

References

1. Lesk AM. Introduction to Protein Architecture (2001, Oxford University Press).
2. Lesk AM. Introduction to Bioinformatics (2002, Oxford University Press)
3. Fersht A. Structure and Mechanism in Protein Science (1999, WH Freeman and Co.)
4. Voet D & Voet JG. Biochemistry (3rd ed., 2003, John Wiley & Sons). Ch.9 (pp.276-319).