Protein Folding and Stability

Protein Folding and StabilityIntroductionProtein folding is the process by which proteins adopt their functional secondary and tertiary structures. Proteins fold to achieve the lowest possible energy state by maximizing favorable interactions, minimizing unfavorable interactions, and maximizing the...

Protein Folding and StabilityIntroductionProtein folding is the process by which proteins adopt their functional secondary and tertiary structures. Proteins fold to achieve the lowest possible energy state by maximizing favorable interactions, minimizing unfavorable interactions, and maximizing the total of the system. Under physiological conditions, proteins experience a when they fold, meaning protein folding is spontaneous.2.3.01 The Hydrophobic EffectFor most proteins, folding involves burying in the protein interior (ie, hydrophobic collapse). This occurs due to a phenomenon called the hydrophobic effect, which is the dominant driving force in most protein folding. Burying the hydrophobic residues has several energetic effects, some favorable and others unfavorable. These effects include:The entropy of the protein decreases. By folding into a specific conformation, the protein is unable to adopt as many states as it could before folding. In other words, the protein becomes more ordered. When considering the protein alone this effect is energetically unfavorable, but this effect is more than offset by the much larger entropy increase experienced by water.The entropy of the water surrounding the protein increases. In unfolded proteins, hydrophobic residues are exposed to water. Water surrounds all molecules that are dissolved in it, forming a shell called a around them. However, water cannot with hydrophobic residues. Consequently, water instead forms hydrogen bonds with the other water molecules in the solvation layer, producing a highly ordered (low entropy) structure. The low entropy of water in this scenario is highly unfavorable.In contrast, water can form strong, enthalpy-releasing interactions with hydrophilic groups. These bonds include and. Consequently, interactions between water and hydrophilic groups are much more favorable than those between water and hydrophobic groups.When a protein folds and the hydrophobic groups are buried, water no longer forms highly ordered solvation layers around those groups. This allows the entropy of the water in the system to increase, which is highly energetically favorable and is the largest contributor to the energy of protein folding. The change in the entropy of water upon protein folding is depicted in Figure 2.24. Chapter 2: Peptides and Proteins64Figure 2.24 The hydrophobic effect hides hydrophobic residues from water, increasing the entropy of the water molecules in the system.Interactions between hydrophobic residues increase. The hydrophobic residues buried within the protein interact with each other through. Although these forces are individually weak, the collective strength of all the London dispersion forces together substantially contributes to the energetic favorability of protein folding.As discussed in Lesson 2.2, interactions between also contribute to the stability of a protein\'s tertiary structure. For example, hydrophilic residues at the surface-interior interface form specific interactions with each other that stabilize the final native conformation (see Figure 2.25). Figure 2.25 Interactions between hydrophilic residues help stabilize native conformation. Chapter 2: Peptides and Proteins65Interactions between two hydrophilic side chains have similar favorability to interactions between a side chain and water, and therefore surface side chains often interact with water instead of each other. Consequently, although interactions between surface side chains do make an important contribution to protein structure, they are not the driving force in protein folding.Concept Check 2.9How would transferring a protein from an aqueous solution to a nonpolar solvent such as hexane most likely affect the folded form of the protein?The influence of the hydrophobic effect gives rise to several classes of proteins: fibrous, globular, and intrinsically disordered. Examples of each are shown in Figure 2.26.Figure 2.26 Depictions of fibrous, globular, and intrinsically disordered proteins.Fibrous proteins are insoluble, often due to significant levels of hydrophobic residues on the protein surface. This is frequently the result of a high overall percentage of hydrophobic residues in the primary structure, which prohibits full burial of hydrophobic regions. Fibrous proteins tend to have a single type or very few types of and very simple, if any, tertiary structure.However, fibrous proteins exhibit significant quaternary structure as multiple identical polypeptides group together, typically into long, insoluble fibers. Each subunit interacts with hydrophobic residues in adjacent subunits, hiding those residues from water. Examples of fibrous proteins include collagen (a major Chapter 2: Peptides and Proteins66component of tendons) and myosin fibers involved in muscle contraction. Fibrous proteins typically provide structure to cells and organs.Globular proteins tend to have a mix of hydrophobic and hydrophilic residues that permits most of the hydrophobic residues to be buried. Consequently, these proteins are typically soluble in water. Globular proteins are perhaps the most common and best-known type of protein and include many enzymes along with proteins that carry out nonenzymatic functions. These proteins may adopt highly complex secondary, tertiary, and quaternary structures.Intrinsically disordered proteins contain a high percentage of hydrophilic amino acids and may also contain significant amounts of proline, which disrupts α-helices. Because these proteins have relatively few hydrophobic residues, they do not undergo hydrophobic collapse and do not adopt notable tertiary structures. Secondary structure is also often absent from these proteins.Many proteins contain some regions that are globular and perform specific functions and other regions that are intrinsically disordered (ie, flexible loop regions) that allow for greater motion within the protein.2.3.02 Energy of Protein FoldingThis concept discusses the energy landscape of protein folding. Although the information in this concept is unlikely to be tested directly on the exam, comprehension of these principles helps understanding of how protein shape is determined and how shape may change in response to changing conditions, which are commonly tested topics.The free energy associated with protein folding is a. Therefore, the native (ie, correctly folded) structure of a protein is the same regardless of the pathway the protein takes to get there. A given protein could follow any of multiple pathways, adopting various intermediate conformations, as it folds.Interestingly, the number of possible conformations a protein could theoretically adopt during folding is so large that, if left to fold by randomly exploring every possibility, a single protein would take longer to fold than the age of the known universe. Yet most proteins fold within a few milliseconds of synthesis.The only explanation for this observation is that proteins do not fold randomly. Instead, local secondary structures tend to form first---amino acid sequences that are conducive to α-helices quickly form α-helices, and those that are conducive to β-strands and turns quickly form β-strands and turns. Note that much of this happens as the protein is being synthesized.As secondary structures come together to form the tertiary structure, the protein may then explore certain possible conformations, and two identical proteins may take different pathways to reach the same final, folded form. Consequently, the energetics of protein folding are often represented by an or funnel as shown in Figure 2.27. Chapter 2: Peptides and Proteins67 Figure 2.27 Example of a protein folding energy landscape, depicting pathways a protein may take during folding.However, the conformations that folding proteins can explore are limited, and most of the theoretically possible conformations are excluded because adopting them would require too much energy input. Each time the protein goes from a high-energy state to a lower-energy state, it tends not to revert to any higher-energy states, allowing the protein to quickly \"funnel\" to the lowest-energy conformation possible.2.3.03 Conformational ChangesAlthough each protein has a lowest-energy conformation, no protein is entirely restricted to this single state. Instead, the atoms in proteins and the bonds between them are constantly moving. Therefore, the protein constantly undergoes small changes in shape, called conformational changes. In other words, proteins are dynamic rather than static.Many proteins have relatively unstructured regions that regularly undergo substantial shape changes. In addition, although the highly structured regions of a protein are less dynamic, they can still undergo noticeable shape changes. Structured regions tend to spend most of their time in or near the lowest-energy conformation possible, but at any given moment a small subset of the proteins in solution may briefly undergo a conformational change to a higher-energy state before rapidly returning to the low-energy conformation (see Figure 2.28). Chapter 2: Peptides and Proteins68Figure 2.28 Depiction of a protein briefly adopting a higher-energy conformation. If conditions remain constant, the protein will quickly return to its low-energy conformation.However, changes in the local environment of a protein may alter its energy landscape. In doing so, a higher-energy conformation may be stabilized and become lower in energy. Similarly, the conformation that was lowest in energy in the initial conditions may increase in energy. Consequently, changes in a protein\'s conditions may induce conformational changes and cause proteins in solution to adopt a new shape (see Figure 2.29). Chapter 2: Peptides and Proteins69Figure 2.29 Changes in conditions may change the energy landscape, resulting in a new lowest-energy conformation.Environmental conditions that can alter a protein\'s most stable conformation (and thereby induce a conformational change) include salt concentration, pH, temperature, and the presence of molecules that bind the protein, called ligands (see Lesson 3.1). Changes in the protein\'s primary structure (eg, through mutation) and chemical alterations, called (Lesson 2.4), can also alter the conformation of the protein. The ability of a protein to change shape in response to environmental conditions is critical to its ability to carry out its biological function.2.3.04 Protein Denaturation and StabilityIn some cases, changes in environmental conditions can alter the thermodynamics of protein folding so much that it becomes more favorable for the protein to lose most or all of its tertiary and secondary structure, rendering the protein nonfunctional. In other words, unfolding becomes spontaneous under certain conditions. When this happens, the protein is said to unfold or denature. Figure 2.30 shows an example of how an energy landscape might change under denaturing conditions. Chapter 2: Peptides and Proteins70Figure 2.30 Under certain conditions, the energy landscape of folding changes enough to favor spontaneous denaturation.Proteins can be denatured by several means, including sufficiently large changes in temperature, pH, salt concentration, or the presence of chemicals known as denaturing agents.TemperatureThe temperature of a system measures the of the molecules within that system; as temperature increases, so does average kinetic energy. In other words, increasing temperature causes the amino acid residues within a protein to move more energetically. The increased motion can cause the intramolecular forces that stabilize protein structure to break (see Figure 2.31), allowing the protein to unfold. Chapter 2: Peptides and Proteins71Figure 2.31 Heat can break intermolecular forces, causing protein denaturation.pHAlthough protein tertiary structure is stabilized primarily by the hydrophobic effect, interactions between hydrophilic surface groups can make a significant contribution to structure. Adjusting the pH can alter the of surface amino acids. For instance, a sufficient increase in pH will deprotonate lysine and arginine residues.Consider a positively charged residue that interacts with a negatively charged residue. Deprotonation of lysine at high pH or protonation of glutamate at low pH may disrupt the interaction. Therefore, pH values that are too high or too low can significantly alter the shape of a protein (see Figure 2.32). Although the hydrophobic core is likely to remain mostly intact regardless of pH, the shape of the protein surface may be altered enough to render the protein nonfunctional.Figure 2.32 Changes in pH interrupt ionic interactions, leading to partial protein denaturation.Salt ConcentrationLike changes in pH, changes in salt concentration may disrupt interactions between charged residues on the protein surface. In this case, the disruption is caused by dissolved salts into positive cations and negative anions. Chapter 2: Peptides and Proteins72A positively charged sodium ion may outcompete arginine or lysine for interactions with glutamate or aspartate, and a negatively charged chloride ion may outcompete glutamate or aspartate for interactions with arginine or lysine. As with pH, altered salt concentration typically has a small or no effect on the hydrophobic core of the protein but disrupts the surface enough to prevent the protein from functioning. Figure 2.33 shows the ions of sodium chloride disrupting an ionic interaction in a protein.Figure 2.33 Salts can disrupt salt bridges in proteins and thereby cause partial denaturation.Denaturing AgentsVarious other chemicals may be added to a protein-containing solution and cause the proteins to denature. These denaturing agents may work by a variety of mechanisms, but most disrupt the hydrophobic effect (see Figure 2.34).For instance, sodium dodecyl sulfate (SDS) contains a long hydrophobic tail that can interact with the hydrophobic residues of a protein, disrupting the hydrophobic core that drives protein folding. The hydrophilic end of SDS can simultaneously interact favorably with water. SDS is commonly used as a denaturing agent in protein electrophoresis (see Chapter 14). Other common denaturing agents include urea and guanidinium chloride. Chapter 2: Peptides and Proteins73Figure 2.34 Example of a denaturing agent (SDS) interacting with the hydrophobic portions of a protein and causing denaturation.Conformational StabilityThe conformational stability of a protein refers to the free energy change between folded and unfolded forms. A protein that undergoes a large, negative free energy change upon folding is more stable than a protein that undergoes a smaller free energy change. Protein denaturation techniques can be used to measure a protein\'s conformational stability.The more stable a protein is in its folded form, the greater the temperature required to denature it. The of a protein is the temperature at which half of the proteins in solution are denatured and half remain folded. Therefore, a more stable protein has a higher melting temperature. Chapter 2: Peptides and Proteins74Concept Check 2.10A researcher measures the percentage of folded protein in a solution both for the wild-type and for a mutated form of a protein of interest. The following graph shows the melting curves of both protein variants. Based on the graph, does the mutation increase or decrease the protein\'s conformational stability?Less commonly, the stability of a protein may be measured by examining the concentration of a denaturing agent required to produce denaturation. In general, if a high concentration of denaturing agent is required to denature a protein, the protein is more stable.2.3.05 Misfolding and ChaperonesMost proteins fold into their native forms. However, some proteins can adopt relatively stable intermediates as they fold. These intermediates correspond to local valleys within the energy landscape. Although such an intermediate is not the lowest-energy conformation possible, the protein may become trapped in this nonfunctional conformation because exiting it requires a high. Proteins that become trapped in stable intermediate conformations are said to be misfolded (Figure 2.35). Most, if not all, cells experience some degree of protein misfolding. Chapter 2: Peptides and Proteins75Figure 2.35 Example of a protein adopting a stable but nonfunctional conformation. The protein may become trapped in this state.Exposure to high temperatures, for example, may cause some proteins to partially or fully denature, as discussed in Concept 2.3.04. Some proteins can refold (ie, renature) once denaturing conditions are removed. However, others cannot renature on their own because they become trapped as relatively stable intermediates before reaching the correctly folded form.Pathology of Protein MisfoldingSome genetic mutations may result in proteins that essentially always misfold. In this case, most of the proteins get degraded and are unable to perform their biological function. This is the basis for a common form of cystic fibrosis.In other cases, misfolded proteins may aggregate (ie, stick to each other). This occurs primarily because misfolded proteins fail to fully bury their hydrophobic residues. Consequently, the exposed hydrophobic residues in one protein may interact with the hydrophobic residues of another, hiding each set of hydrophobic residues from water (Figure 2.36). Therefore, aggregation shares some features with quaternary structure in that multiple polypeptides interact, often with hydrophobic residues at their interface. However, because the individual polypeptides and their aggregates are not the native, functional form of the protein, aggregation commonly has negative biological consequences. Chapter 2: Peptides and Proteins76Figure 2.36 Example of protein aggregation.Protein aggregates may take various forms. In some cases, multiple misfolded proteins assemble into highly ordered fibrous structures known as. These fibers are associated with various neurodegenerative disorders, including Alzheimer disease. Other proteins may form much less ordered, amorphous aggregates. These aggregates have also been implicated in certain disease states, including cataract formation.Certain types of misfolded proteins can act as infectious agents that can be transmitted from one organism to another. These proteins, known as , can induce correctly folded proteins to misfold and become prions themselves. This can lead to a catastrophic cascade effect because increasing amounts of misfolded protein induce increasing amounts of correctly folded protein to misfold. Prions are the underlying cause of Creutzfeldt-Jakob disease and its transmissible variant, mad cow disease.Concept Check 2.11Identify the labeled groups (ie, 1, 2, 3) in the following diagram as a correctly folded protein, an amyloid fibril, or a prion:ChaperonesAn entire class of proteins, called chaperones, exist to aid in the correct folding of other proteins. Chaperones are sometimes also called heat shock proteins (HSPs) because they help prevent cellular damage due to high temperatures. Chaperones provide both newly synthesized and misfolded proteins with an environment that facilitates proper folding. This often involves interactions between hydrophobic residues in the chaperone and hydrophobic residues in the protein to be folded. These interactions protect the hydrophobic residues from water and thereby prevent aggregation while the protein is folding into its proper form.In addition to helping newly synthesized proteins fold correctly, chaperones can also help misfolded proteins to adopt the correct conformation. Many chaperones help protein aggregates to disaggregate and then help the monomers to fold correctly. This is depicted in Figure 2.37