Question

7. Discuss the primary issues associated with protein folding.

Answer 1

The “protein folding problem” consists of three closely related puzzles: (a) What is the folding code? (b) What is the folding mechanism? (c) Can we predict the native structure of a protein from its amino acid sequence? Once regarded as a grand challenge, protein folding has seen great progress in recent years. Now, foldable proteins and nonbiological polymers are being designed routinely and moving toward successful applications. The structures of small proteins are now often well predicted by computer methods. And, there is now a testable explanation for how a protein can fold so quickly: A protein solves its large global optimization problem as a series of smaller local optimization problems, growing and assembling the native structure from peptide fragments, local structures first.

1. The Folding Code

The starting point of protein folding is indeed the primary structure (the sequence of amino acids), also known as denatured state of the protein. Even the smallest amount of the denatured state can activate nucleation and proliferation carried out through protein folding pathways. Characterization of these denatured states of proteins at physiological conditions is very difficult because it is necessary to unfold the proteins to their denatured states without the presence of denaturants [2, Travagilini-Allocatelli et al.].

Recent research has allowed the study of denatured states to reach new heights using the single-molecule approach. Researchers used single-molecule experiments to examine coil to globule transition of proteins and have demonstrated that the denatured state showed steady expansion as the concentration of denaturant was increased. Similarly, at low denaturant concentrations, the peptide chain of the protein collapsed in a sequence dependent manner [2, Travagilini-Allocatelli et al.].

Also there have been advancements to study intermediates in protein folding. For example, the denatured state of the engrailed homeodomian (En-HD) was engineered to be denatured in physiological conditions and Nuclear Magnetic Resonance (NMR) has shown that it resembles a folding intermediate. An additional study discovered that the specific section of the En-HD called the helix-turn-helix motif (HTH) behaves as an independent folding domain. When examining the full protein, the HTH motif represents a folding intermediate in the En-HD folding pathway [2, Travagilini-Allocatelli et al.].

2. Structure Prediction

Nowadays, researchers predict the structure of a protein by inputting the amino acid sequence into a computer. The advanced technology and modeling software allow scientists and researchers to form a predicted structure. However, the structure is not accurate, as there is always a small degree of errors present. Nevertheless, this can speed up discovery of new medications since the digital structure can be manipulated.

Secondary structure prediction

Secondary structure prediction is a set of techniques that aim to predict the secondary structures of proteins and RNA sequences based only on their primary structure which is amino acid or nucleotide sequence. For example, proteins, a prediction consists of assigning regions of the amino acid sequence as alpha helices, beta strands, or turns. The success of a prediction is determined by comparing it to the results of the DSSP (the DSSP algorithm is the standard method for assigning secondary structure to the amino acids of a protein, given the atomic-resolution coordinates of the protein) algorithm applied to the crystal structure of the protein; for nucleic acids, it may be determined from the hydrogen bonding pattern. Specialized algorithms have been developed for the detection of specific well defined patterns such as transmembrane helices and coiled coils in proteins, or microRNA structures in RNA.

Tertiary structure prediction

Experimental methods such as NMR spectroscopy or x-ray diffraction analysis are widely used in order to determine tertiary protein structures. But the rate at which protein structures can be determined by experimental techniques is much lower than the rate at which new genes are identified by the various genome projects.

3. Folding Speed and Mechanism

This is also known as the Levinthal's paradox. Nowadays, we have advanced methods such as mutational methods, which give us the value of phi and psi during folding, and hydrogen exchange methods, which allow us to see structural folding events. However, the dynamics and mechanism of protein folding still require additional research and understanding.

The dynamics and kinetics of unfolded polypeptide chain have been addressed by recent studies of loop formation by Keifhaber and coworkers. They used different model systems each representing different types of loops: end to end, end to interior, or interior to interior. Their experiments showed that end to interior and interior to interior loop formation formed slower than end to end loops. This discovery suggests that chain motion of one part of the unfolded polypeptide chain is coupled to other parts of the chain. These kinetics experiments also revealed that protein folding processes take place on different time scales and thus there is a hierarchy in loop formation[2, Travagilini-Allocatelli et al.].

Hope this helps