Chapter 5: The Three Dimensional Structure of Proteins

What does Anfinsen's experiment tell us about protein folding? Why could this conclusion be made? What was this experiment?

Anfinsen's experiment tells us that the information required for protein folding is founded in the protein's primary structure and that the process is spontaneous. Many hundreds to thousands of similar experience have been run on many different proteins which all led to the same basic conclusion: The final structure of a protein is determined by the primary structure and this process is exergonic, favoring the folded structure. It is a spontaneous reaction. It also shows that all the information that is needed to fold a protein is found in the primary structure. Another conclusion is that protein folding is accurate, specific, and reproducible. His experiment is: He would add a chemical (urea or BME) to denature a protein, then would remove the chemical, and then the protein would go back to its native state.

What does Levinthal's paradox tell us about protein folding?

Levinthal's paradox tells us that protein folding is not simply a random search through all possible conformations but rather a direct process. Assumption 1: 100 amino acid proteins can be made in about 5 seconds Assumption 2: Each protein can form only 10^100 conformations -> 10 phi and 10 psi angles for each amino acid -> gross underestimate Assumption 3: A protein conformation can be sampled ever 10^-13 seconds -> again, this is an arbitrary number -> probably an overestimate Results: It would take 10^77 years to search through all of the possible conformations Conclusion: Protein folding must follow a predetermined path, it is spontaneous, folding is accurate, specific, and reproducible

What are the phi and psi angles? What is a Ramachandran plot? How is it generated? What is the least restricted amino acid? What is the most restricted amino acid?

The phi angle = alphaN-alphaC The psi angle = alphaC - C Each bond can rotate in a 360 degree circle and thus these bond angles have values, by convention, ranging from -180 degree to +180 degrees. Each alphaC has both a phi and a psi angle. The Rakmachandran plot is a means of visualizing the phi/psi angle combinations of every amino acid within a protein. The psi angle (Y axis) is plotted agains the phi angle (X axis). Densely populated ares of the plot are referred to as the "allowed" regions - regions where the phi/psi angle combinations result in a minimum of steric clashes between atoms and is thus thermodynamically favored. The sparsely populated regions are the disallowed regions. The most conformationally restrictive amino acid is proline with its side chain linked to the amino acid.

What is quaternary structure? Is it found in all proteins? What is a subunit? How are subunits held together?

The quaternary structure defines the 3D spatial arrangements of each subunit has with respect to one another. Not all proteins have a quaternary structure- those are said to operate as a monomer. A subunit is a single polypeptide chain that interacts with additional polypeptide chains via non covalent interactions to form a multimeric complex. Subunits are held together by non covalent interactions.

What is the primary structure? Will a proteins primary structure always consist of 1 chain?

The sequence of amino acids contains the information necessary to determine the 3D structure of the protein and thus is the first or primary level of structure within a protein. A proteins primary structure will not always consist of one chain (insulin as an example has two chains), but most will.

What is a protein folding funnel? Why is this used rather than a reaction coordinate diagram?

The vertical axis of the funnel represents the proteins internal energy whereas the horizontal axis of these "funnels" represents the number of conformations at that particular energy value. It is used rather than a coordinate diagram because it allows for a great number of beginning conformations that will "fold" as they go down the funnel to the most thermodynamically favorable conformation.

What is a globular protein? What is a long-range interaction? What types of bonds can be long-range interactions?

A globular protein is a protein that consists of helices and other secondary structural elements compact into a tight glob like shape. Long range interactions are when electrostatic interactions are formed between residues far away from each other in the primary structure but close together in the tertiary structure. Long range interactions can be covalent (such as disulfide bonds) and non covalent. Globular proteins are usually found in aqueous environments.

What is a motif? How does it differ from a domain?

A motif is a super-secondary structure, or protein fold, is the structural pattern within a protein that will consist of two ore more secondary structure elements, their connecting loops, as well as their 3D relationship with respect to one another. A domain is defined as a portion of a polypeptide that is larger than a super-secondary structure that is both stable and functionally independent of the rest of the protein. They are different in that motifs are not functionally and structurally independent of the protein.

What is a beta turn? What are loops within protein structure?

A beta turn is a structural element that creates a complete reversal of the polypeptide chains. They consist of exactly 4 amino acids in an antiparallel sheet. Protein loops are a much broader range of "turns" within a polypeptide chain. They are well defined structural elements in any given protein.

What is the role of a chaperone? What is an amyloid fibril?

A chaperone is a protein that assists any other protein in adopting its final structure while not become a part of that structure. An amyloid fibril is a misfolding as a result of intermolecular interactions between beta sheets which as been thought to play a roll in the progression of a number of diseases including Alzheimers, Parkinson, transmissible spongiform encephalopathy, Huntingtons, and type 2 diabetes. In most cases, a soluble protein that is normally secreted from the cell is secreted in a misfolded state and converted into an insoluble extracellular amyloid fiber. The diseases are collectively referred to as amyloidoese.

What does it mean for the peptide bond to have partial double bond character? How does this restrict this bond? Is the peptide bond cis or trans in proteins? Are there exceptions? What types of noncovalent interactions would you expect the peptide backbone to participate in?

A peptide has a double bond partial character because it shares an electron in the peptide bond between the oxygen and the nitrogen. This partial double bond of the peptide bond results in the rigid and planar properties of the peptide bond. The peptide bond in proteins is most often the trans configuration. The exception to this rule is when the amino acid in the first position is followed by a proline in the second position. In this case, the peptide bond adopts the cis configuration about 20 to 25% of the time due to the likely occurrence of resulting steric clashes in either configuration, make them essentially energetically equivalent. Another non covalent interaction that a peptide backbone participates in is due to the permanent dipole, which exists with its positive end at the amend hydrogen and the negative end at the carbonyl oxygen. With this dipole, the peptide backbone can participate in hydrogen bonds, which are partially responsible for stabilizing several levels of the protein structure.

What are prosthetic groups? How do these relate to function?

A prosthetic group is a nonprotein molecule that is permanently associated with a protein and is crucial for its function. This relates to function because proteins interact with a wide variety of molecules within the cellular environment and the interactions are mediated by the structure and electrostatic complementarity between the protein and the molecules it interacts with.

What are the features of a beta sheet? What is the difference between a strand and a sheet? How do parallel and anti-parallel strands differ? Which is more stable?

Beta strand features: pleated appearance, side chains will alternate from one side of the strand to the other side of the strand Beta sheets are comprised of two or more extended polypeptide chains commonly referred to as beta-strands. Beta-sheet features: pleated appearance, side chain alteration from 1 side to the other, hydrogen bonding (between the amide groups and the carbonyl group), antiparallel (more stable) vs parallel - both of which are found in nature, idealized torsion angles, connections between strands can be 100's of amino acids long or they can be as short as 4 amino acids long In parallel sheets each strand runs in the same direction where the beginning of the strand is the side closest to the N-terminus and the end of the strand is closes to the C-terminus. Anti-parallel sheets are the opposite. Parallel strands have phi angles equal to -113 degrees and psi angles equal to +113 degrees. Anti-parallel sheets have phi angles of -139 degrees and psi angles equal to +135 degrees. Antiparallel sheets have geometry where the hydrogen bond donor points directly at the hydrogen bond acceptor. In parallel sheets, the hydrogen bond geometry is less than ideal. Because of this, if all else is equal, antiparallel strands will be more stable than the parallel strands.

What are the features of an alpha helix? How is it stabilized? If the backbone did not participate in any non-covalent interactions in the primary structure would the formation of the helix have a negative or positive change in enthalpy? Why is this a simplified statement?

Features of the alpha helix are: left or right handed, 5.4 angstrom pitch (the elevation gained for 1 complete turn of the helix), 3.6 residue per turn, R groups point away from the center of the helical axis, R groups are offset from one another progressing down helix, hydrogen bonding that exists between carbonyl of the nth residue and the amide of the n + 4 residue, and ideal phi angle of -57 degrees and psi angle of -47 degrees It is stabilized by the hydrogen bonds within the backbone and in the R groups. All of the carbonyl groups are pointing up and all of the amino groups are pointing down, perfectly lined up for hydrogen bonding. Glycine and proline are destabilizing. Every third to 4th residue will interact because they are on the same side of the helix. If the backbone did not participate in any non-covalent interactions in the primary structure, ??????????? An idealized right handed alpha helix will have phi angles equal to -57 degrees and psi angles equal to -47 degrees. Any stretch of peptide backbone that adopts these phi/psi angles will naturally lead to the development of structural features above.

What is the sign on the change in enthalpy for a protein when it goes from the unfolded to the folded state? Why is this sign difficult to quantitate? In other words why is this debated? In general, what would a good argument be for the fact that this sign should be negative (think environment)?

It is hard to determine the sign of the change in enthalpy for a protein when it goes from the unfolded state to the folded state. This is difficult to quantitate because although there is a large number of bonds (hydrogen and ionic for instance in myoglobin) being formed when the protein folds there are many bonds being broken between the unfolded state and the surrounding solvent, which consist of water capable of interacting via hydrogen bonds with the protein. Disulfide bonds as well as ionic interactions in the hydrophobic core will favor a negative delta H.

What are the major forces that stabilize and destabilize protein structure? This is one you need to think about. For instance what would happen if you started mutating different residues within a protein? How would this stabilize or destabilize the structure? Think about steric clashes introduced by going from a small to a large amino group. Think about like charges repelling and the introduction of a polar group in the hydrophobic core. How would pH affect these interactions? What about temperature?

Major forces that stabilize a protein are hydrophobic effects, hydrogen bonds, ionic bonds, van Der Waal forces, and disulfide bonds. Things that destabilize a protein are unfavorable interactions like solvent-exposed hydrophobic side chains, electrostatic repulsion resulting from, for example, the placement of like charges in too-close proximity, phi/psi angle combinations in the disallowed region of the Rakmachandran plot, and steric clashes. If you started mutating different residues within a protein, it could destabilize the protein. If you added a bigger amino acid, you might get steric clashes. You could also loose the amphipathic features of the protein. If you introduced a polar group into the hydrophobic core, the core would begin to become hydrophilic. pH could change these interactions by changing the dipoles within the amino acid by protonating or deprotonating different portions of the R group on the amino acids, which could change the way it interacts with other amino acids. Also changing temperature could change the change the overall change in free energy for proton folding and thus affect the stability of the protein. Things that can make delta H negative (making delta G more negative, meaning more stable thermodynamically) -> disulfide bridges -> salt bridges -> buried salt bridges, recall coloumbs law where the bond strength is determined by the surroundings and the proximity of the ions

What is the sequence of atoms within the peptide backbone?

N_alpha is the alpha-amino group, C_alpha is the alpha-carbon group, and C is the carbon of the carbonyl. The peptide backbone is the peptide minus atoms that are found in the side chains.

What is secondary structure? What is a recurring pattern?

Secondary structure are the reoccurring backbone patterns of which there are 4 primary types: alpha-helix, beta-sheet, beta-turns, and loops.

What is the overall sign on the change in entropy for a protein when it goes from the unfolded to the folded state? What contributes to this entropy term? What are the individual signs on these contributions?

The change in enthalpy is positive when a protein goes from a folded to an unfolded state. When the protein itself folds, the protein undergoes a reduction in enthalpy because it goes from a less conformationally rigid form to a more rigid form. However, we must include the change in entropy of the solvent too. Upon folding, the hydrophobic core is formed, allowing each water molecule to return to bulk solvent and a disordered state. The solvent goes a drastic increase in entropy and therefore exhibits a positive change in entropy which greatly outweighs the negative change in entropy of the protein folding itself. As a result most biochemists agree that the formation of hydrophobic interactions drives the folding process toward the native state and is the major contributor to the overall stability of a protein.

Why does a salt bridge on the interior of a protein have a greater strength than those on the surface?

The dielectric constant found on the interior of a protein is estimated to be about 4 as compared to the dielectric constant of water at 78.4. The strength of an ionic bond is inversely related to the dielectric constant of the surrounding medium making the ionic bonds within a protein much stronger than those on the surface.

The "hydrophobic core" is a term biochemist often use to talk about the interior of a protein. What does this mean?

The hydrophobic core of a protein is the interior of a globular protein that consists primarily of hydrophobic side chains. The hydrophobic residues are basically hiding from the water.