Protein Folding PDF

# Protein Folding - Process in which a polypeptide chain goes from a linear chain of amino acids with a vast number of more or less random conformations in solution to the native, folded tertiary (and for multichain proteins, quaternary) structure ## Introduction and Protein Structure - Proteins...

# Protein Folding - Process in which a polypeptide chain goes from a linear chain of amino acids with a vast number of more or less random conformations in solution to the native, folded tertiary (and for multichain proteins, quaternary) structure ## Introduction and Protein Structure - Proteins have several layers of structure, each of which is important in the process of protein folding. - The first, most basic level of this structure is the sequence of amino acids themselves (**primary structure**). - The next layer in protein structure is the **secondary structure**. Secondary structure includes α-helices and ß-sheets. - The **tertiary structure** is the next layer in protein structure. This takes the α-helices and ß-sheets and allows them to fold into a three-dimensional structure. ## Why do proteins fold? In order to carry out their function (for instance as enzymes or antibodies), proteins must take on a particular shape, also known as a "fold". Thus, proteins are truly amazing machines: before they do their work, they assemble themselves! This self-assembly is called "folding". - **From genome:** ACU UUC CGU AAC - **To protein sequence:** THR PHE ARG ASN **Forms determines function** Suppose you have some molten iron. You may turn it into nails, hammers, wrenches, etc. What makes these tools different from each other is their **form**, i.e. their shape and structure. ## Protein Folding - Proteins are folded and held together by several forms of **molecular interactions**. The molecular interactions include the **thermodynamic stability** of the complex, the **hydrophobic interactions**, and the **disulfide bonds** formed in the proteins. ## Protein Folding - Protein folding considers the question of how the process of protein folding occurs, i.e. **How the unfolded protein adopts the native state?** - It has been aptly been described as the second half of the **genetic code.** - Predict 3D structure from primary sequence - Avoid misfolding related to human disease - Design protein with novel function ## Protein Folding Depends Upon - The Process depends upon: - The solvent (water of lipid bilayer) - The concentration of salt - The PH - The temperature - The possible presence of cofactor - Molecular chaperones ## Process of Protein Folding - **Primary Structure** The primary structure of a protein, its linear amino-acid sequence, **determines its native conformation**. The specific amino acid residues and their **position** in the polypeptide chain are the determining factors for which portions of the protein fold close together and form its three-dimensional conformation. **The amino acid composition is not as important as the sequence.** ## Secondary Structure - Formation of a secondary structure is the **first step** in the folding process that a protein takes to assume its native structure. - Characteristic of secondary structure are the structures known as alpha helices and beta sheets that fold rapidly because they are stabilized by **intramolecular hydrogen bonds**, as was first characterized by Linus Pauling. - Formation of intramolecular hydrogen bonds provides another important contribution to protein stability. - Protein secondary structure takes on the three forms: - Alpha helix - Beta sheet - Turn, coil or loop ## Hydrogen Bonding Scheme for Alpha Helix Main chain oxygen of ith residue (written as O(i)) Amide of N (i+4)th residue (written as N(i+4)) O(i)-----N(i+4) e.g. 1---5,2-----6-- - O(i)-----N(i+4) e.g. 1---5,2-----6-- - -- ## Tertiary Structure - The alpha helices and beta pleated sheets can be **amphipathic** in nature or contain a hydrophilic portion and a hydrophobic portion. This property of secondary structures aids in the tertiary structure of a protein in which the folding occurs so that the hydrophilic sides are facing the aqueous environment surrounding the protein and the hydrophobic sides are facing the hydrophobic core of the protein. ## Protein Structure - 1. Peptide bond - 2. Disulfide bridge - 3. Hydrogen bond - 4. Ionic bond - 5. Hydrophobic Interactions ## Quaternary Structure - Tertiary structure may give way to the formation of quaternary structure in some proteins, which usually involves the **"assembly" or "co assembly"** of subunits that have already folded; in other words, multiple polypeptide chains could interact to form a fully functional quaternary protein. ## Driving Force of Protein Folding - Folding is a **spontaneous** process that is mainly guided by **hydrophobic interactions**, formation of intramolecular hydrogen bonds, van der Waals forces, and it is opposed by conformational entropy. The process of folding often begins **co-translationally**, so that the N-terminus of the protein begins to fold while the C-terminal portion of the protein is still being synthesized by the ribosome; however, a protein molecule may fold spontaneously **during or after biosynthesis**. ## Hydrophobic Effect - Protein folding must be **thermodynamically favorable** within a cell in order for it to be a spontaneous reaction. Since it's known that protein folding is a spontaneous reaction, then it must assume a **negative Gibbs free energy value**. - **Minimizing the number of hydrophobic side-chains exposed to water** is an important driving force behind the folding process. - The hydrophobic effect is the phenomenon in which the hydrophobic chains of a protein collapse into the **core of the protein** (away from the hydrophilic environment). - The multitude of hydrophobic groups interacting within the **core of the globular folded protein** contributes a significant amount to protein stability after folding, because of the **vastly accumulated vander Waals forces** (specifically London Dispersion forces). ## Chaperones - A class of proteins that **aid in the correct folding** of other proteins in vivo. Chaperones exist in all cellular compartments and interact with the polypeptide chain in order to allow the **native three-dimensional conformation** of the protein to form; however, **chaperones themselves are not included in the final structure of the protein they are assisting in.** ## Molecular Chaperones Assist Protein Folding: - **Definition:** In molecular biology, Chaperones are proteins that **assist covalent folding or unfolding**, and **assembly or disassembly** of other macromolecular structures. - They bind to **unfolded and partially folded polypeptide chains** to prevent the improper association of exposed hydrophobic segments that might lead to non-native folding as well as polypeptide aggregation and precipitation. ## Molecular Chaperones - In Molecular Biology, molecular chaperones are proteins that assist the covalent **folding or unfolding** and the **assembly or disassembly** of other macromolecular structures. - Chaperones are present when the macromolecules perform their **normal biological functions** and have correctly completed the processes of **folding and/or assembly**. - Chaperones may assist in folding even when the **nascent polypeptide is being synthesized** by the ribosome. - Molecular chaperones **operate by binding to stabilize** an otherwise unstable structure of a protein in its folding pathway. - They assist the de novo folding of proteins or they form **repair machines** for **misfolded or even aggregated** proteins, and they are therefore especially important for the **survival of cells during stress situations**. ## A Well Studied Example: - A well studied example is the **bacterial GroEL system**, which assists in the **folding of globular proteins**. - In **eukaryotic organisms**, chaperones are known as **heat shock proteins**. - These are basically proteins that are involved in the **folding and unfolding** of other proteins. - Various approaches have been applied to study the **structure, dynamics and functioning** of chaperones. ## Chaperone-Assisted Protein Folding - Chaperone-assisted folding is required in the **crowded intracellular environment** to prevent aggregation. - Used to prevent **misfolding and aggregation**, which may occur as a consequence of exposure to **heat or other changes** in the cellular environment. ## Chaperones - There are two major classes of molecular chaperones in both prokaryotes and eukaryotes: - The **Hsp70 family** of 70-kD proteins - the **chaperonins** (large multisubunit proteins) ## Hsp70 Proteins: - An Hsp70 protein binds to a newly synthesized polypeptide. - The Hsp70 chaperone probably helps prevent premature folding. ## Chaperon Protein Folding Process - **Unfolded substrate proteins bind** to a hydrophobic binding patch on the interior rim of the open cavity of GroEL. - **Binding of substrate protein** in this manner, in addition to binding of ATP, induces a conformational change that allows association of the binary complex with a separate lid structure, GroES. - **ATP is hydrolyzed and releases the GroES**, which promote folding of protein. ## Anfinsen Experiment - **Native ribonuclease** (Diagram of native ribonuclease with amino acid residues labeled) - **Denatured reduced ribonuclease** (Diagram of denatured ribonuclease with amino acid residues labeled) - **Denaturation of ribonuclease A** (4 disulfide bonds), with 8 M Urea containing b-mercaptoethanol, leads to random coil and no activity. ## Anfinsen Experiment - **Denatured reduced ribonuclease** (Diagram of denatured ribonuclease) - **Dialysis to remove urea and b-mercaptoethanol** - **Air oxidation of the sulfhydryl groups in reduced ribonuclease** - **Native ribonuclease** (Diagram of native ribonuclease with amino acid residues labeled) - **After renaturation, the refolded protein has native activity, despite 105 ways to renature the protein.** - **Conclusion**: All the information necessary for folding into its native structure is contained in the amino acid sequence of the protein. ## Anfinsen Experiment - **Scrambled ribonuclease** (Diagram of scrambled ribonuclease with amino acid residues labeled) - **Remove b-mercaptoethanol only**, oxidation of the sulfhydryl group, then remove urea - **scrambled protein, no activity** - **Further addition of trace amounts** of b-mercaptoethanol converts the scrambled form into native form. - **Conclusion**: The native form of a protein has the **thermodynamically most stable structure** ## The Levinthal Paradox - The Levinthal Paradox states that the number of possible conformation available to a protein is astronomically large. - Imagine a 100-residue protein so it has 99 peptide bonds so 198 phi and psi angles if each of these bond angles can be one of 3 stable conformation so maximum 3198 occur different conformations so require a long time than the age of universe to arise at its correct native conformation. - **Conclusion**: folding not a random, but have specific path way. ## Experimental Techniques for Studying Protein Folding: - X-ray crystallography - Fluorescence spectroscopy - Circular Dichroism ## X-Ray Crystallography - Crystallography is one of the more efficient and important methods for attempting to decipher the three-dimensional configuration of a folded protein. - To be able to conduct X-ray crystallography, the protein under investigation **must be located inside a crystal lattice.** - Only by relating the **electron density clouds with the amplitude of the x-rays** can this pattern be read and lead to assumptions of the phases or phase angles involved that complicate this method. ## Fluorescence Spectroscopy - Fluorescence spectroscopy is a highly sensitive method for studying the folding state of proteins. - Three amino acids, phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), have intrinsic fluorescence properties, but only Tyr and Trp are used experimentally because their **quantum yields are high enough** to give good fluorescence signals. ## Protein Folding Models - **Framework Model:** - This suggests that folding is start with formation of secondary structure which then interact to form a more advanced folding intermediate. - **Framework Model Diagram:** (Framework model diagram with N and C termini) - Supported by experimental observation of rapid formation of secondary structure during protein folding process. - **Framework Model Diagram:** (Framework model diagram with N and C termini) - Formation of individual secondary structure elements ## Molten Globule State - The molten globule state is an intermediate conformational state between the native and fully unfold states of globular protein. - Some character of molten state: - The presence of native-like content of secondary structure. - The absence of specific tertiary structure produced by the tight packing of amino acid side chains. - Model for early stages of protein folding (hydrophobic collapse) ## Collapse Model of Protein Folding - During this intermediate stage, the native-like elements are thought to take the form of sub-domains that are not yet properly docked to form domains. - In the final stage of folding, the protein undergoes a series of complex motions in which it attains its relatively rigid internal side chain packing and hydrogen bonding while it expels the remaining water molecules from its hydrophobic core. ## Nucleation Condensation Model - In this model, the secondary and tertiary structure at a time made, the hydrophobic care collapse in random fashion and form a native structure. - **Nucleation Condensation Model Diagram:** (Nucleation Condensation Model diagram with N and C termini) - **Nucleation Condensation Model Diagram:** - Formation of a nucleus of hydrophobic residues (Nucleation Condensation Model diagram with N and C termini) - **Expansion of nucleus** (Nucleation Condensation Model diagram with N and C termini) ## Protein Folding Mechanism - **Nascent protein:** nonfunctional, linear - **Native:** functional, Non linear 3D **Linear Amino Acid Strand** (Diagram of amino acid chain with a funnel leading to 3D conformation) - **Bumps in Funnel represent energy barriers** and the folding will take the path of least resistance to its final native state. - **Native 3D Conformation** (Diagram of 3D conformation) ## Protein Folding - Protein folding is either by: - **Co translational process** (N-terminus is folded while the C-terminus is synthesizing) - **After translation** ## Incorrect Protein Folding - A protein is considered to be misfolded if it cannot achieve its normal native state. This can be due to **mutations** in the amino acid sequence or a disruption of the normal folding process by external factors. - Misfolded protein typically contains ß-sheets that are organized in a supramolecular arrangement known as a **cross-ß structure.** These ß-sheet-rich assemblies are very stable, very **insoluble**, and generally resistant to proteolysis. - The misfolding of proteins can trigger the further misfolding and accumulation of other proteins into aggregates or oligomers. The increased levels of aggregated proteins in the cell leads to formation of **amyloid-like structures**, which can cause degenerative disorders and cell death. ## Disease Caused by Misfolding - Alzheimer's disease - Cystic fibrosis - Parkinson's disease - Huntington's disease - Gaucher's disease ## Unfolding of Proteins - **Denaturation:** - **Introduction**: Denaturation is a process in which a protein **loses its native shape** due to the **disruption of weak chemical bonds** and interactions, thereby becoming biologically **inactive**. ## For Example - Changing **pH** denatures proteins. - Certain reagents such as **urea and guanidine hydrochloride** denature proteins. - Detergents such as **sodium dodecyl sulphate** denature proteins by associating with **non-polar groups** of proteins. ## Denatured Protein - When protein is denatured, it loses its function. - **Examples:** - A denatured enzyme ceases/stops its function. - A denatured antibody does not bind to its antigen. - The denatured state of protein does not necessarily mean that **complete unfolding or denaturation** of protein. - Under some conditions, these proteins exhibit both properties: **denaturation and renaturation.** ## Mechanism of Protein Unfolding - Unfolding of native proteins occur at both temperatures: **higher temperature and lower temperature**. - **Types of Denaturation:** - **Heat Denaturation/Thermal Denaturation** - **Cold Denaturation.** ## How Denaturation Occurs at the Level of Protein Structure - Denaturation occurs at the secondary, tertiary, and quaternary structure **but not at the primary structure level**. - When the shape is compromised and the molecule **can no longer function in its desired capacity**. ## Final Note - Interactions between the side chains of amino acids determine how a long polypeptide chain folds into the intricate three-dimensional shape of the functional protein. As a peptide folds, its amino acid side chains are attracted and repulsed according to their chemical properties. For example, positively and negatively charged side chains attract each other. Conversely, similarly charged side chains repel each other. In addition, interactions involving hydrogen bonds, hydrophobic interactions, and disulfide bonds all exert an influence on the folding process. This process of trial and errors tests many, but not all, possible configurations seeking a compromise in which attractions outweigh repulsions. This results in a correctly folded protein with a low-energy state.