Lectures 3, 4, 5: Chapter 4: 3-D structure of proteins

What is a dihedral (torsion) angle? What are the important dihedral angles in a peptide and what do they rotate around?

1) A dihedral angle is a angle at the intersection of 2 planes. 2) IN polypeptides, there are 3 bond vectors connection 4 main chain atoms. The three angles (vectors) are: a) Phi (circle with cross through it): C-N-C alpha-C bonds (rotation about the N-C alpha bond). b) Psi(devils dagger): N-Calpha-C-N bonds (rotation about Calpha-C bond). c) Omega (w): C alpha-C-N-Calpha bonds (rotation about C-N bond). *NOTE: This bond is in trans position constrained , almost never rotates.

What is a domain? What are the main characteristics of domains within polypeptides? In a protein with multiple domains, how does each domain appear? What are some examples of domains in pyruvate kinase? Show how they look in the picture.

1) A domain is a part of a polypeptide that is independently stable or could undergo movements as a single entity with respect to the entire protein. 2) Main characteristics: a) Polypeptides with more than a few hundred amino acid residues often fold into two or more domains, often with different functions. b) A domain from a large protein will maintaind their 3-dimensional structure when seperated from the remainder of the polypeptide chain. 3) In a protein with multiple domains, each domain appears as a distinct globular lobe. 4) Some examples are nucleotide binding domain, substrate binding domain, and regulatory domain.

What is a motif and why is it useful? What are some examples? Draw them

1) A motif (also known as a fold or supersecondary structure) is a recognizable folding pattern involving two or more elements of secondary structure and the connections between them. It is useful because complex structures are built from motifs/ 2) Examples of motifs include beta-alpha-beta loop, coiled coil, helix bundle, beta hairpin, greek key, helix-turn-helix. Further explanation: A motif can be very simple, such as two elements of secondary structure folded against each other, and represent only a small part of a protein. An example is abeta-alpha-beta loop. A motif can also be large, such as the helix bundle or the beta barrel. Note that a motif is not a hierarchical structural element falling between secondary and tertiary structure. It is simply a folding pattern. The synonymous term "supersecondary structure" is thus somewhat misleading because it suggests hierarchy.

Are alpha helices right handed or left handed? How do you determine if it is right or left handed from looking at the structure?

1) Alpha helices are always right handed. 2) There is a simple method for determining whether a helical structure is right-handed or left-handed. Make fists of your two hands with thumbs outstretched and pointing away from you. Looking at your right hand, think of a helix spiraling up your right thumb in the direction in which the other four fingers are curled as shown (clockwise). The resulting helix is right-handed. Your left hand will demonstrate a left-handed helix, which rotates in the counterclockwise direction as it spirals up your thumb.

What are beta turns, what do they connect, and what do the turns involve? Why do Gly and Pro residues often occur in beta turns? Where are the two most common forms of beta turns found and why?

1) Beta turns link successive runs of alpha-helix or beta conformation. Beta turns are turns that connect the ends of two adjacent segments of an antiparallel beta sheet. The structure is a 180 degree turn involving four amino acid residues, with the carbonyl oxygen of the first residue forming a hydrogen bond with the amino-group hydrogen of the fourth. 2) Gly is small and flexible, making it easy to occur in beta turn. -Pro occurs in beta turns ecause peptide bonds involving the imino nitrogen of proline readily assumes the cis configuration. 3) The two most common forms of beta turns are found near the surface of the protein because it is where the peptide groups of the central two amino acid residues in the turn can hydrogen-bond with water.

What is the change in gibbs energy seperating the folded and unfolded states in typical proteins and why? What are the factors that favor unfolding?

1) Change in G seperating the folded and unfolded states in typical proteins is small, so about 20 to 65 kj/mol. -It is small because there are factors that favor unfolding and folding, so there is marginal stability from one to the other. 2) Factors that favor unfolding: a) Unfolded proteins have ability to assume countless confirmations, there is more range in the unfolded state, so the unfolded state is high in conformational entropy. b) Many groups in unfolded polypeptide chain have a lot of hydrogen-bonding interactions with the solvent (water), maintaining the unfolded protein structure.

What is circular dichroism spectroscopy and what causes it? How does it relate to alpha helix and beta conformation?

1) Circular dichroism spectroscopy is how we measure secondary structure of proteins. It measures the difference in absorption of left-handed and right-handed plane-polarized light. This difference is caused by structural asymmetry in a molecule. It also helps with looking at how proteins unfold. 2) The alpha helix and beta conformations have characteristic circular dichroism spectroscopy.

Where is collagen found? How is the secondary structure of collagen arranged and what is the amino acid sequence of it?

1) Collagen is found in connective tissue (tendons, cartilage), the organic matrix of bone, and the cornea of the eye. 2) How it is arranged: a) A single collagen helice (NOT alpha helix, quite distinct from it) is arranged in a left handed helix with 3 amino acid residues/turn. It is a repeating tripeptide unit of Gly-X-Y (where X is typically Pro and Y is typically 4-hydroxyproline). b) Three seperate collagen helices (called alpha-chains) are supertwisted about each other in a right-handed supertwist.

Why is Glycine, Pro, and 4-hyp important in collagen structure? How many variants of collagen do mammals have?

1) Gly is in the center, between individual alpha-chains, to form tight junctions, for tight packing. -Pro and 4-Hyp permit sharp twists of collagen helix, so for tight twists. 2) Mammals have over 30 structural variants of collagen.

How is the polypeptide arranged in a beta conformation? What is a beta sheet? How do hydrogen bonds form? How are the R groups arranged? What is the difference between a parallel and antiparallel beta sheet?

1) In beta conformation, the backbone of the polypeptide chain is a zigzag structure (rather than helical). See fig a. 2) A beta sheet is the arrangement of several segments side by side in beta conformation. 3) Hydrogen bonds form between adjacent segments of polypeptide chain within the sheet (figure b). 4) The R groups of adjacent amino acids protrude from the zigzag structure in opposite directions, creating alternating patterns (fig a). -AKA R groups of B-conformations extend above and below the strand in an alternating manner perpendicular to the backbone carbonyl oxygens and amino hydrogens. 5) See fig b, c. Parallel beta sheets have the same amino to carboxyl orientations, and antiparallel have opposite amino to carboxyl orientations.

What is the structure of the alpha helix? What are the repeating units?

1) In this structure, the polypeptide backbone is tightly wound around an imaginary axis drawn longitudinally through the middle of the helix, and the R groups of the amino acid residues protrude outward from the helical backbone. 2) The repeating unit is a single turn of the helix.

What does the Raachandran plot show? Draw/show a positive rotation on phi and a positive rotation on psi.

1) It shows the allowed phi/psi angles.

Poll question: You are working with a protein that has glutamate at position 80, which is in the middle of a long alpha helix. Given the structure of a right-handed alpha helix, predict which amino acid (s) at position 84 would stabilize the helical structure and why? Options: Alanine, Lysine, Aspartate, Glutamate, and threonine.

1) Lysine, because glutamate hs negatively charged carboxyl group in R side chain, so to compliment, lysine has positively charge amine group in R side chain.

According to the Ramachandran plot, where do most values of phi and psi fall taken from known protein structures fall? What amino acid is found outside these regions and why?

1) Most values of phi and psi taken from known protein structures fall into the expected regions, with high concentrations near the alpha helix and beta conformation values as predicted. 2) The only amino acid residue often found in a conformation outside these regions is glycine. Because its side chain is small, a Gly residue can take part in many conformations that are sterically forbidden for other amino acids.

If a polypeptide has a long block of Glu residues, will this segment of chain form an alpha helix at pH 7.0, and why or why not? If a polypeptide chain has many adjacent Lys and Arg residues at pH 7, will and alpha helix form and why? Based on the twist of the alpha helix, what are the critical interactions of the amino acid side chain? If polypeptide chain has Asn and Ser adjacent to eachother, does alpha helix form and why? Why do proline and glycine have the least proclivity to form the alpha helix? What terminal are negatively charged amino acid residues found on alpha helical segment, and are + charged amino acid residues found?

1) No, negatively charged carboxyl group of adjacent Glu residues repel eachother so strongly that they prevent the formation of the alpha helix. 2) No, the positively charged R groups of Lys and Arg repel eachother and prevent formation of the alpha helix. 3) See pic attached, figure 44 D. Between an amino acid side chain and the side chain that is 3 (or 4) residues away on either side are the most critical interactions. The positively charged amino acid residues are often found 3 residues away from the negatively charged amino acid residues, forming and ion pair. 4) Asn and Ser do not form alpha helix because the bulkiness of the shape destabilizes the alpha helix. 5) Proline: In proline, the nitrogen atom is part of a rigid ring, and the rotation about the N-C (alpha) bond is not possible. This introduces a destabilizing kink in an alpha helix. Also, the Nitrogen atom of Proline residue in a peptide linkage has no Hydrogen to participate in H bonds with other residues. -Glycine: glycine has more conformational flexibility, and polymers of glycine take up coiled strcutures that are different. 6) Negatively-charged amino acids found near the N-terminal (positive dipole end) and positively charged amino acid residues are found near the C-terminal.

When is phi and psi +/- 180 degrees? Draw a polypeptide connecting 2 amino acids and indicate the psi and phi angles are at 180 degrees. What happens to the psi angle as the 4th atom rotates clockwise relative to the first? WHen are the first and 4th atoms eclipsed? Do this on molecular model set.

1) Phi and psi are +/- 180 when the polypeptide is fully extended and all the peptide groups are in the same plane. This is when the first and 4th atoms are furthest apart. 2) As the 4th atom rotates clockwise relative to the first, the phi and psi angles increase. From the +/180 position, the dihedral angle increases from -180 to 0, nd 0 is when the first and 4th atoms are eclipesd. 3) https://www.youtube.com/watch?v=Kewhg5spUjs -video explains

What allows for a large number of confirmations in proteins? What form do most isolated proteins exist in and why? What determines the 3-D structure of a protein? How are

1) Proteins consist of a large number of individual bonds. Because of this, free rotation is possible around the individual bonds.And because of all of the free rotation, there is a very large number of possible confirmations (which is any structural state a protein can achieve without breaking a covalent bond). 2) Despite the large number of possible confirmations, most proteins exist in one or a few predominant confirmations. This is because of stability. The predominant confirmations under biological conditions, also known as the native state, are usually the ones that are thermodynamicaly most stable, when gibbs free energy is at the minimum. 3) 3-D structure (confirmation) of a protein is determined by the amino acid sequence.

What is Quaternary structure? What functions does the association of multiple subunits within a protein serve?

1) Quatenary structure describes the interaction of multiple subunits (ie polypeptide chains) within a protein structure. 2) The association of multiple subunits within proteins can serve a variety of functions including: a) Regulatory role: The binding of small molecules can affect the interaction between subunits, causing a change in proteins response (function) to small changes in concentration in regulatory molecules. b) Structural role: Some associations, like fibrous proteins and coat proteins of viruses, serve primary structural roles. c) Very large protein samples participate in complex multistep reactions.

What does SCOP database stand for and what are the 4 classes of protein structure it uses? What is a protein family?

1) SCOP database stands for Structural Classification of Proteins. The four classes of proteins are all alpha, all beta, alpha/beta (alpha and beta are interspersed or alternating), and alpha + beta (alpha and beta regions are somewhat segregated). 2) Protein families are groups of proteins with significant structural similarity.

What does secondary structure mean? how do dihedral angles work in regular secondary structures? What are the most common types of secondary structure?

1) Secondary structure is when any CHOSEN SEGMENT of the polypeptide chain, describing LOCAL spatial arrangement of the MAIN CHAIN atoms (ignoring side chains etc). 2) Secondary structures occur when each dihedral angle phi and psi remains the same or nearly the same throughout the segment. 3) The most common secondary structures are alpha helices and beta.

Look at the photo and state which ones are parallel and which ones are antiparallel.

1) See photo

What are the two main rules of structural patterns? Poll question: Which amino acid would most likely be found in interior of globular (compact) protein? Ala, D, Glutamate, lysine, and cysteine.

1) Structural patterns: Two patterns: a) Hydrophobic residues are buried in protein interior, away from water. b) Number of Hydrogen bonds and ionic interactions within the protein is maximized. 2) Alanine because it is the most hydrophobic in the amino acids in this list.

What is tertiary structure? How are interacting segments of polypeptide chains held in tertiary positions?

1) Tertiary structure is the overall 3D arrangement of all atoms in a protein. It includes longer-range aspects of amino acid sequence. So amino acids that are far apart in the polypeptide sequence (which may have different secondary structures) can interact within the completely folded 3-D structure of a protein. 2) Several kinds of weak interactions (and sometimes covalent bonds) between segments hold the chains in place.

How are the alpha carbons of adjacent amino acid residues separated and how are they arranged? Show this using glycine and valine. What did X-ray diffraction studies of polypeptides show and what does this indicate? Draw resonance structures. How do the 6 atoms of the peptide group lie, and draw this using alanine and valine? Can the peptide C-N bonds rotate freely, and why or why not? Where is roatation permitted? Draw diagram showing this using glycine and valine.

1) The alpha carbons of adjacent amino acid residues are separated by three covalent bonds, arranged as C (alpha)—C—N—C (alpha). 2) X-ray diffraction studies show that the peptide C-N bond is SHORTER than the C-N bond in simple amines. It also shows that atoms associated with peptide bonds are COPLANAR, indicating the resonance or partial sharing of the two pairs of electrons between the carbonyl oxygen and amide nitrogen (Oxygen has partial negative charge and hydrogen bonded to nitrogen has partial + charge, so there is a small electric dipole). 3) The 6 atoms lie in a single plane, with oxygen of carbonyl group TRANS to the hydrogen of the amide nitrogen. 4) Peptide C—N bonds, because of their partial double-bond character, cannot rotate freely. Rotation is permitted about the N—C(alpha) and the C(alpha)—C bonds. The backbone of a polypeptide chain can thus be pictured as a series of rigid planes, with consecutive planes sharing a common point of rotation at C(alpha) (Fig. 4-2b). The rigid peptide bonds limit the range of conformations possible for a polypeptide chain.

What is alpha-keratin found in? How are alpha-helices of keratin arranged and what does this look like? What are the surfaces of two alpha-keratin helices made up of and what does this permit? What amino acid residues is alpha-keratin rich in?

1) The alpha-keratins have evolved for strength. They are found only in mammals, and consist of almost the entire dry weight of hair, wool, nails, claws, quills, horns, hooves, and much of the outer layer of skin. 2) How alpha-keratin arranged: a) A single alpha-keratin helix (individual polypeptide strands) are arranged in a right-handed alpha-helix. b) Two strands of alpha-keratin are arranged in a parallel (with amino termini at same end) and are wrapped around each other to form a coiled coil, amplifying the strength (like rope). This is a left handed coiled coil. c) Coiled coils combine to form higher ordered structures like protofilaments and protofibrils. 3) The surfaces where the two alpha-helices touch are made up of hydrophobic amino acid residues, their R groups meshed together in a regular interlocking pattern. This permits a close packing of the polypeptide chains within the left-handed supertwist. 4) Not surprisingly, alpaha-keratin is rich in the hydrophobic residues Ala, Val, Leu, Ile, Met, and Phe.

How do we use secondary structure to determine tertiary structure?

1) The arrangement of polar and non-polar side chains on a secondary structure element (alpha-helix of beta-strand) gives an indication of its position and orientation in the tertiary structure.

When discussion tertiary and quartenary structure, what are the two structural groups? What is difference between the two and explain each?

1) The to groups are fibrous proteins and globular proteins, and they differ in both structure and function. 2) Fibrous proteins: -Polypeptide chains arranged in long insoluble strands or sheets, usually of a single repeating unit of secondary structure. -They largely consist of a single type of secondary structure assembled into tertiary/quartenary structures, making the tertiary structure relatively simple. -Insoluble in water because of hydrophobic amino acid residues on interior and exterior of protein. -Function: Provide structural support, shape, external protection, and/or flexibility. b) Globular proteins: -Polypeptide chains folded into a spherical or globular shape. -Typically consist of several types of secondary structure. -Function: Most enzymes and regulatory proteins are globular proteins. -More compact than fibrous proteins.

Why does the alpha-helix form more readily than many other possible confirmations? Can alpha-helices form in polypeptides consisting of D and L amino acids and what is most stable form of alpha helix consisting of D amino acids? What is the overall alpha helix dipole?

1) Why alpha helix forms more readily: a) It is stabilized by the Hydrogen bond between the hydrogen atom (attached to the Nitrogen atom that links the peptides) and the Oxygen (carbonyl oxygen of 4th amino acid on amino terminal side of peptide bond). See figure 4.4a. -Basically, between the carbonyl O negative and the amide H positive, creating a strong dipole moment in peptide bond. b) Alpha helix is also more stabilized by Hydrogen bonds between residues in nearby peptide chain. 2) All residues must be of one stereoisomeric series; a D-amino acid will disrupt a regular structure consisting of L-amino acids, and vice versa. The most stable form of an alpha helix consisting of D-amino acids is left-handed. 3) The large macroscopic dipole moment is from the O negative of carbonyl to the H positive (of amide).

In principle, phi and psi can have any value between -180 and +180, but why are many values prohibited? How do allowed values of phi and psi become evident, and how do you plot it? In a Ramachandran plot, what does dark blue, medium blue, light blue, and white represent? What does asymmetry of the plot result from? What has a broader range, glycine or proline, and why? What do steric clashes determine?

1)Many values are prohibited by steric interference between atoms in the polypeptide backbone and amino acid side chains. 2) Allowed values for phi and psi become evident when psi is plotted versus phi in a Ramachandran plot. 3) Dark blue is when there is no steric overlap, medium blue is when there is a slightly unfavorable steric clash, and light blue is only permissable with omega dihedral flexibility. 4) The asymmetry of the plot results from the L stereochemistry of the amino acid residues. The plots for other L residues with unbranched side chains are nearly identical. 5) The Gly residue, which is less sterically hindered, has a much broader range of allowed conformations. The range for Pro residues is greatly restricted because phi is limited by the cyclic side chain to the range of -35 to -85. 6) Steric clashes determine high energy of phi/psi combinations.

What are the factors that favor folding? Explain each.

Factors that affect folding: 1) Hydrophobic interactions resulting from the shielding of hydrophobic residues from water. Explanation: -Pure water has H20 hydrogen-bonded molecules. Other molecules in water disrupt the hydrogen bonding of water. -When water surrounds a hydrophobic molecule, the optimal arrangement of H20 hydrogen bonds results in a highly structured shell (solvation layer) surrounding the molecule. -This highly ordered H20 molecules in solvent leads to a decrease and unfavorable entropy. -However,, when nonpolar groups CLUSTER, the solvation layer extent decreases (because nonpolar groups are now clustered, not unfolded, so less to surround), leading to a favorable increase in entropy. -THE INCREASE IN ENTROPY IS THE DRIVING FORCE for association for hydrophobic groups in aqueous solution. So hydrophobic amino acid side chains cluster (because more favorable for water) in the protein interior, away from the water. -This Hydrophobic effect is the main driving force in protein folding. -So amino acid sequence of most proteins have a significant amount of hydrophobic amino acid side chains, which are positioned so they are clustered when protein is folded, forming a hydrophobic protein core. 2) Hydrogen bond interactions between proximal groups on protein. Explanation: -Polar groups can generally form hydrogen bonds with water, and are soluble in water. But Hydrogen bond/unit mass is greater for pure water than any other liquid or solution, so there are limits to solubility even in the most polar molecules (because the presence of polar molecules causes a decrease in hydrogen bonds/unit mass). -So a solvation layer to some extent forms aroundpolar molecules. -And the energy of formation of Hydrogen bonds between proximal groups of protein are usualy canceled because of interactions between the groups and water, but the release of structured water as the hydrogen bonds/intamolecular interactions form provides entropic driving force for folding. 3) Ionic interactions between nearby charged groups on the polypeptide: Explain: -Interaction of oppositely charged groups that form an ion pair can have a stabilizing or destabilizing effect on protein structure. -When UNFOLDED, in the case of hydrogen bonds, charged amino acid side chains interact with water and salts, and the loss of those (ionic) interactions due to interactions with water must be considered. -SO the strength of the ionic interaction...ASK about dielectric constan. 4) Disulfide (covalent) bond formation Explain: -Van der wals interactions are dipole-dipole interactions. -As atoms approach each other, these dipole-dipole interactions provide an attractive intermolecular force. -Individual, they are weak, but in a protein that is well-packed, or in an interaction between two proteins, the number of van der wals interactions is substantial, causing proteins to fold.

What are the five constraints affecting the stability of an alpha-helix?

Five constraints: 1) Intrinsic property of an amino acid residue to form an alpha helix (table 4.2). 2) Position of amino acid residue relative to its neighbour. Interactions between amino acid side chains can stabilize or destabilize the alpha helix. Particularly the amino acid side chains that are space 3 (or 4) residues apart. 3) The bulkiness of adjacent R groups. 4) The occurrence of Pro and Gly residues, which have the least proclivity to form alpha helices. 5) Identities of amino acid residues near the ends of the alpha-helical segment of the polypeptide. The partial positive charge of the net helix dipole of polypeptide on the peptide amino group (N-terminal). and partial negative charge is on carbonyl group (C-terminal). As a result, negatively-charged amino acids found near the N-terminal (positive dipole end) and positively charged amino acid residues are found near the C-terminal.

What are the rules that gave emerged from common protein folding patterns?

See picture