Protein Folding: Sequence, Structure, and Pathway

The fundamental question of how the amino acid sequence of a protein determines its unique three-dimensional structure (conformation) is central to biochemistry. The native structure is the most thermodynamically stable form. 

1. Determining Secondary Structure

Secondary structures are local, regularly repeating conformations like the α-helix and β-strand.

·    Amino Acid Preferences: Certain amino acid residues have preferences for specific secondary structures, revealed by their frequency of occurrence in known proteins.

• α-Helix Promoters: Alanine, Glutamate, Leucine.
• β-Strand Promoters: Valine, Isoleucine.
• Turn/Loop Promoters: Glycine, Asparagine, Proline. 

·    Steric and Structural Constraints: These preferences are based on the compatibility of the residue's side chain with the local geometry:

• Steric Clash: Residues with branching at the β-carbon (Val, Thr, Ile) destabilize α-helices due to clashes but are easily accommodated in β-strands.
• Competing H-Bonds: Serine and Asparagine often disrupt α-helices because their polar side chains compete for main-chain hydrogen bonding groups.
• Proline: Disrupts both α-helices and β-strands because it lacks a main-chain NH group (essential for H-bonding) and its cyclic structure severely restricts its geometry.
• Glycine: Its high flexibility makes it well-suited for tight reverse turns. 

·   Prediction Difficulty: Predicting secondary structure based only on local sequence preferences is difficult (only 60–70% accurate) because:

• Preferences are marginal (e.g., Glutamate prefers α-helix by only a factor of three).
• Tertiary Interactions (interactions between residues far apart in sequence) are often decisive in stabilizing the final structure of a segment.
• Context is crucial; the environment provided by the rest of the protein is essential. 

2. Protein Folding as a Cooperative Transition

o Denaturation and Unfolding: Proteins can be denatured (unfolded) by disrupting the weak non-covalent bonds that stabilize the structure using heat or chemical agents (e.g., urea). 

o  "All or None" Process: The transition between the folded (native) and unfolded (denatured) states, observed when changing denaturant concentration, is sharp, suggesting an "all or none" or cooperative transition.

• Cooperative Folding Rationale: The stability of one part of the structure depends on interactions with the rest of the protein. If one region begins to unfold, the loss of its stabilizing interactions destabilizes the surrounding structure, leading to a rapid, complete unraveling of the entire protein. 

o    Molecular Reality: Although the solution appears to be a mixture of only fully folded and fully unfolded molecules (a two-state model) at the transition midpoint, protein folding cannot occur in a single step. Unstable, transient intermediate structures must exist between the two states. 

3. The Folding Paradox and Pathway

·      Levinthal's Paradox: A random, exhaustive search of all possible conformations for even a small 100-residue protein would take astronomically long (1.6 x 10²⁷ years). Since small proteins fold in milliseconds, they cannot sample every conformation.

This paradox proves that folding must follow a defined pathway with intermediates. 

·       Cumulative Selection: The solution to the paradox is that proteins fold by the principle of cumulative selection—partly correct intermediate structures are retained (stabilized by free energy) and used as a foundation for further folding. 

·       Nucleation-Condensation Model (Energy Funnel):

• Mechanism: Local regions with strong structural preferences rapidly form an initial structure (nucleation), which then guides and stabilizes the formation of additional structure (condensation). 

• Energy Funnel: The folding process can be visualized as moving down an energy funnel. The wide rim represents the high-energy, unfolded state with many conformations. As the protein folds, free energy decreases, and the accessible conformational space narrows, leading to the bottom: the low-energy, unique native state. 

4. Exceptions to the Single-Structure Paradigm

While the general paradigm is that a sequence specifies one structure, exceptions exist:

o    Intrinsically Unstructured Proteins (IUPs): These proteins (or regions, often rich in charged/polar residues) lack a discrete 3D structure under physiological conditions. They assume a defined structure only upon interacting with a specific molecular partner. This versatility allows a single protein to interact with multiple partners, performing different functions (especially in signaling). 

o    Metamorphic Proteins: These proteins exist as an ensemble of two or more distinct structures (conformations) that are in equilibrium and have approximately equal energy.

• Example: The chemokine lymphotactin exists in equilibrium between a receptor-activating chemokine structure and a β-sheet dimer structure that binds carbohydrates. Both distinct structures have mutually exclusive but required functions. 

5. Approaches to Structure Prediction

Predicting the native 3D structure from the amino acid sequence is an unsolved challenge, with two main approaches:

·         1. Ab Initio Prediction (From the Beginning):

• Attempts to calculate the minimum free energy structure or simulate the folding process without prior knowledge of similar structures.
• Limited by the sheer number of possible conformations and the subtle, marginal stability of proteins.

·         2. Knowledge-Based Methods:

• Examines an unknown sequence for compatibility with known protein structures or fragments in databases (e.g., using sequence homology).
• If a significant match is found, the known structure serves as a reliable initial model (template) for the unknown protein.