Protein Folding

Protein folding is a fundamental process in molecular biology, crucial for proteins to attain their functional forms and perform specific biological tasks. Protein folding in structural biology can help explain how a protein's amino acid sequence correlates with its three-dimensional shape, which in turn determines its function. Protein misfolding causes diseases ranging from neurodegenerative conditions like Alzheimer's to Parkinson's. Understanding the intricacies of protein folding not only deepens our knowledge of cellular processes, but also aids in the development of therapeutic strategies to combat diseases associated with protein misfolding.

Figure 1: Folded, 3-D structure of ribonuclease A.

Mechanisms of Protein Folding

Proteins fold through a complex process driven by physical and chemical interactions within the polypeptide chain. The central dogma of structural biology suggests that a protein's primary sequence—its linear amino acid sequence—contains all the information needed to determine its final, functional structure. Proteins fold to minimize free energy, forming stable structures with specific arrangements of secondary, tertiary, and sometimes quaternary elements.

The Anfinsen's Dogma or the thermodynamic hypothesis proposes that a protein will naturally fold into the conformation with the lowest possible free energy, which is its native structure. This theory, based on experiments by Christian Anfinsen, suggests that protein folding is a spontaneous process dictated by the primary sequence. The funnel-shaped energy landscape model further refines this idea, illustrating how proteins fold by gradually reducing their free energy as they move toward a stable, native state.

Figure 2: The diagram sketches how proteins fold into their native structures by minimizing their free energy.

Proteins typically fold through multiple intermediate states, which are often characterized as folding intermediates. These intermediates serve as waypoints along the folding pathway and can prevent misfolding or aggregation. In structural biology, techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy and time-resolved X-ray crystallography help visualize these intermediates, offering insight into folding pathways and transition states that are difficult to observe directly.

Figure 3: Real-time NMR timescale and use of the real-time NMR method to understand the protein folding/unfolding process. (Bhattacharya et al., 2023)

Levels of Protein Structure in Folding

Protein folding involves the arrangement of amino acids into hierarchical structural levels to create a stable and functional molecule. The process is stepwise. The primary structure is the linear sequence of amino acids, while the secondary structure refers to localized folding patterns such as alpha helices and beta sheets that are stabilized by hydrogen bonds. In the tertiary structure, secondary elements pack together to form a three-dimensional configuration, creating a stable core in which hydrophobic residues are usually buried. Finally, the quaternary structure occurs when multiple polypeptide chains or subunits assemble to form a functional protein complex.

Figure 4: The four levels of protein structural architecture. (Bhattacharya et al., 2023)

The folding process itself depends on non-covalent interactions, including hydrogen bonds, van der Waals forces, hydrophobic effects, and electrostatic interactions. Hydrophobic residues typically move toward the protein's core, minimizing their exposure to water, while hydrophilic residues are more likely to be exposed on the surface, interacting with the aqueous environment. This arrangement ensures stability and correct orientation for functional interactions.

Figure 5: Graphical representations of common non-covalent interaction types. These include hydrogen bonds, salt bridges, van-der-Waals forces, π-cation interactions, sandwich or π-π stacking or T stacking. (Aranda-Garcia et al., 2022)

Factors Influencing Protein Folding

Several factors influence the protein folding process, including the amino acid sequence, the cellular environment, and the presence of molecular chaperones:

Amino acid properties, such as hydrophobicity, charge, and size, play a significant role in determining folding patterns, influencing whether specific residues are exposed or buried within the core.

The cellular environment also profoundly affects folding. High concentrations of other macromolecules create a crowded environment, which can limit available space and alter the folding kinetics, leading to a phenomenon known as macromolecular crowding. In such conditions, proteins may fold differently than in dilute solutions, leading to faster or alternative folding pathways.

Molecular chaperones are specialized proteins that assist other proteins in folding correctly. Chaperones bind to nascent or misfolded proteins, preventing aggregation and guiding them toward their native conformations. Examples of chaperones include the heat shock proteins (HSPs) and chaperonins, which provide an isolated environment for folding, reducing the risk of misfolding and aggregation. The action of chaperones is especially vital under stress conditions, such as high temperatures, where proteins are more prone to unfolding and aggregation.

Figure 6: The HSP70 chaperone cycle. HSP70 is switched between high- and low-affinity states for unfolded and partially folded protein by ATP binding and hydrolysis. Unfolded and partially folded substrate, exposing hydrophobic peptide segments, is delivered to ATP-bound HSP70 (open) by one of several HSP40 cofactors. The hydrolysis of ATP, which is accelerated by HSP40, results in closing of the α-helical lid of the peptide-binding domain (yellow) and tight binding of substrate by HSP70 (closed). Dissociation of ADP catalyzed by one of several nucleotide-exchange factors (NEFs) is required for recycling. Opening of the α-helical lid, induced by ATP binding, results in substrate release. Data Bank (PDB) accession codes 1DKG, 1DKZ, 2KHO and 2QXL. Pi, inorganic phosphate. (Hartl et al., 2011)

Protein Misfolding and Its Implications

Protein misfolding occurs when proteins don't fold into their proper shapes, exposing hydrophobic regions that can cause them to aggregation. These aggregates are usually insoluble and can interfere with essential cellular functions, which can disrupt cellular functions. Misfolded proteins are associated with various diseases, especially neurodegenerative disorders like Alzheimer's and Parkinson's.

Figure 7: Protein misfolding and disease. A conformational change in a normal protein seems to be the hallmark event in a group of diverse diseases. Protein misfolding may be associated to disease by either the absence of biological activity of the folded protein or by a gain of toxic activity by the misfolded protein. Aggregation of the misfolded protein may also contribute to the disease pathogenesis. (Soto, 2001)

Amyloid disorders—Alzheimer's, Parkinson's, Huntington's—are caused by misfolded protein molecular aggregates in the brain. In Alzheimer's, the beta-amyloid peptide misfolds and forms plaques, which are toxic to neurons. And in Parkinson's, alpha-synuclein protein misfolds and forms Lewy bodies, leading to neuronal damage. These diseases illustrate how critical proper protein folding is to maintaining cellular health, as even minor misfolding can lead to significant dysfunction.

To counter misfolding, cells possess quality control mechanisms, including proteasomes and autophagy pathways, which degrade misfolded proteins. The ubiquitin-proteasome system tags defective proteins for degradation, preventing their accumulation. Understanding these pathways in structural biology has facilitated the development of therapies targeting misfolded proteins.

Techniques for Studying Protein Folding

Structural biology offers a variety of tools to explore protein folding:

X-ray crystallography is a powerful method for determining the high-resolution structures of proteins, providing detailed images of folded proteins and their atomic arrangements. Although it typically captures only the final folded state, it helps identify structural motifs and domains critical for stability and function.

NMR spectroscopy takes a different approach, studying proteins in solution, and providing information on folding dynamics and intermediate states. NMR's ability to capture proteins in their near-native environments is particularly valuable for observing folding kinetics and conformational changes, making it essential for studying small proteins and folding pathways in real time.

Cryo-EM, on the other hand, is used for examining larger protein complexes. Though less commonly used for single small proteins, cryo-EM can reveal conformational changes in multi-protein complexes and folding intermediates in larger assemblies, such as molecular chaperones. Computational methods, including molecular dynamics simulations, further complement experimental techniques by predicting folding pathways and structural transitions, offering a detailed view of folding energy landscapes and potential misfolding pathways.

Advances in Structural Biology and Protein Folding Research

Structural biology has seen remarkable advancements in recent years, thanks to innovative technologies that offer deeper insights into protein folding. Modern single-molecule techniques, like optical tweezers (Figure. 8) and single-molecule FRET (Förster resonance energy transfer) (Figure. 9), allow researchers to observe folding in real time at the single-molecule level. This provides a level of detail that traditional bulk methods simply can't achieve, revealing precise folding pathways and dynamic processes. In addition, advances in computational tools and molecular dynamics simulations have expanded our ability to study folding mechanisms. Together, these cutting-edge approaches are opening new doors to understanding how proteins fold, paving the way for breakthroughs in research and applications.

Figure 8: Optical tweezers in single-molecule biophysics. Forces acting on a dielectric sphere interacting with light, with the incident light beam focused by a high-numerical aperture (NA) lens. a | A Rayleigh particle smaller than the wavelength of light experiences a scattering force (F_scat, red arrow) that pushes the particle along the direction of propagation of the light and a gradient force (F_grad, black arrow) that attracts it towards the focus. b | A dielectric sphere larger than the wavelength of light either reflects or refracts light (pink arrows) focused by a high-NA lens. The change in direction of each ray corresponds to a change in momentum of the light and an equal and opposite change in bead momentum. Reflected rays of light lose forward momentum that is gained by the bead, leading to a net force (Freflection, red arrow) pushing the bead along the direction of propagation of the light. Refracted rays are deflected forward because of the high incidence angle of the light, which generates momentum change and reactive force (Frefraction, black arrow) that pulls the bead towards the focus. (Bustamante et al., 2021)

Figure 9: (A) Diagrammatic sketch of the concept of Förster resonance energy transfer; (B) Correlation between FRET efficiency and the distance. Single-molecule Förster resonance energy transfer (smFRET) inherits the strategy of measurement from the effective "spectroscopic ruler" FRET and can be utilized to observe molecular behaviors with relatively high throughput at nanometer scale. (Qing et al., 2021)

In conclusion, protein folding is a complex but highly organized process that is essential for proper protein function. In structural biology, understanding the mechanisms and pathways of protein folding is crucial, as it provides insights into how proteins adopt their functional conformations and how misfolding can lead to diseases.

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References

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2. Bhattacharya S, Gupta S, Hazra S. Nuclear magnetic resonance spectroscopy for protein structure, folding, and dynamics. In: Advanced Spectroscopic Methods to Study Biomolecular Structure and Dynamics. Elsevier; 2023:105-123.
3. Bustamante CJ, Chemla YR, Liu S, Wang MD. Optical tweezers in single-molecule biophysics. Nat Rev Methods Primers. 2021;1(1):1-29.
4. Hartl FU, Bracher A, Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis. Nature. 2011;475(7356):324-332.
5. Qiao Y, Luo Y, Long N, Xing Y, Tu J. Single-molecular förster resonance energy transfer measurement on structures and interactions of biomolecules. Micromachines. 2021;12(5):492.
6. Soto C. Protein misfolding and disease; protein refolding and therapy. FEBS Letters. 2001;498(2-3):204-207.