BIOCHEMISTRY - DR. JAKUBOWSKI
03/20/2009
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Learning Goals/Objectives for Chapter 2C: After class and this reading, students will be able to
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In contrast to micelles and bilayers, which are composed of aggregates of single and double chain amphphiles, proteins are covalent polymers of 20 different amino acids, which fold, to a first approximation, in a thermodynamically spontaneous process into a single unique conformation, theoretically at a global energy minimum. This chapter section will investigate the possible conformations available to proteins, just as we studied the conformations of free fatty acids and acyl chains in lipid aggregates. The next chapter section will discuss the actual processes of folding and of unfolding (denaturation), both in vitro and in vivo. Then we will discuss the thermodynamics and intermolecular forces which stabilize the folded (or native) shape and the unfolded (or denatured state) of proteins, in a fashion similar to how we discussed micelle and bilayer stability.
Main Chain Conformations - Cis/Trans Peptide Bonds/ Ramachandran Plots
Just as saturated fatty acid chains have preferred conformations (all ttt), peptide chains also have preferred conformations. The complexity is much greater, however. With fatty acid chains, we dealt only with torsion or dihedral angles around the methylene carbons. For proteins, we must consider the covalent links which attach the amino acids together, as well as the rotations possible in 20 different amino acids. The peptide bonds connects the carbonyl C of the i th amino acid to the alpha amine N of the i th+1 amino acid. The resulting bond is an amide link. X-ray analysis shows that the the C-N bond has double bond character. This can be accounted for by delocalizing the nonbonding electron pair of the N to the carbonyl C forming a double bond, with the pi bonded electrons of the carbonyl C-O bond moving to the O. These resonance structures lead to a planar arrangement of the peptide carbonyl C and amide N and the two other atoms connected to each, since the hybridization of the C and N has sp2 character, with 120o bond angles. This greatly simplifies the number of conformations which a protein can adopt since these 6 atoms can be considered to reside and move in a plane. The alpha C serves as the corner attachment point of two different planes, each which can rotate independently of the other plane. The two planes can twist around the alpha carbon. The rotation angles for the two planes are called phi (f) and psi (y) are analogous to the torsion angles in the acyl chains of fatty acid. They can vary from -180 to +180o. The R group substituent attached to the alpha C can also rotate around the alpha C and the beta C of the side chain. This angle is defined as chi.. Other rotations also occur within the side chain. We will concentrate on phi (f) and psi (y) angles in this section.
Figure: Extended Polypeptide Showing Planes and phi/psi Angles
Another important feature of the peptide bond is that the alpha Cs at opposite ends of the rectangle are usually trans to each other (on opposite sides of the C-N bond in the peptide bond. This trans arrangement of the alpha Cs is sterically favored by a factor of 1000/1 for all peptide bonds except X-Pro. Pro, which is a cyclic amino acid, is sterically restricted.
Figure: trans arrangement of the alpha Cs
The figure above, which also shows the X-Pro bond, clearly shows that both the trans and cis forms of the X-Pro bonds are hindered to a similar extent. In X-Pro bonds in proteins, the trans/cis ratio found in proteins is 4/1. The diagram above shows rans peptide bonds, and how they could be converted to cis through rotation around the C-N bond.
A protein can now be thought of as a series of linked sequences of rigid, planar peptide units which can rotate around phi/psi angles. When the chain is fully extended (as shown in the links above), phi/psi are 180o.
When phi (f) and psi (y) equal 0o, the two peptide bonds flanking the alpha Cs are in the same plane. This conformation is prohibited since the O of the C=O on one plane and the H of the H-N on the other are overlapping - i.e. they approach closer than their van der Waals radii.
This simple example shows that all conformational space is not accessible for protein folding.
A Web Tutorial - phi/psi Angles . This is an , interactive tutorial that displays two planes connected to an alpha carbon. You can rotate each plane independently, and pick any phi/psi angle and see what the planes look like. It requires Cosmo Player (available on the SGI network) or as a Windows Download from the National Institute of Standards and Technology
Ramachandran was the first to calculate which phi/psi angles are allowed. He modeled the angles permitted to a tripeptide, assuming the atoms were hard spheres. The angles allowed depended in part on the limiting distance chosen for interatomic contacts. (i.e. the usual H -- H distance is 2.0 angstroms, and 3.0 for C--C bonds.) The plot below show the allowed regions in red. Only 3 small regions of conformational space are available. If you allow a closer approach by 0.1 angstrom, more conformation space is available, but only one new area is available (shown in yellow in the plot below).
Figure: Ramachandran plot
A Ramachandran plot of Ala-Ala-Ala is nearly identical to the plot for Phe-Phe-Phe (which is unbranched at the beta carbon (the first methylene C in the side chain). The plot for Thr-Thr-Thr, which has a branch at the beta C (with OH and CH3 attached) shows somewhat less room than the other plots. Pro-Pro-Pro is most restricted for obvious reasons. For a longer chain than a tripeptide, there are more restrictions than for (Ala)3, since the chain can't assume a conformation when it passes through itself. The plots for actual proteins have many points which do fall in forbidden regions. However, these points would be allowed if the peptide bonds is twisted a few degrees. Gly bonds also fall outside the allowed regions. This is understandable, since the side chain of Gly is H, and it is used in protein where sharp turns of the chain is necessary. Right hand alpha helices fall at -57,-47 while left hand alpha helices fall at +57,+47. (Notice these are not mirror images of each other. The mirror image of a right-handed alpha helix would be a left handed helix made of D-amino acids.) Parallel beta sheets are at -119, -113, while antiparallel sheets falls at -139, +135. Other types of helices also are found. The 310 helix , a sharper helix with 3 amino acids/turn, falls at -49,-26. All of these examples of secondary structure fall in allowed regions. Modern Ramachandran plots do not model the atoms as hard spheres but instead consider the potential energy of the atoms using the Lennard-Jones potential (6-12 potential) for van der Waals interactions. We discussed this potential function in the molecular modeling lab.
Figure: Ramachandran plots showing phi/psi angles for Gly, Ala, Tyr, and Pro in actual proteins
Secondary Structure
Secondary structures are those repetitive structures involving H bond between amide H and carbonyl O in- the main chain. These include alpha helices, beta strands (sheets) and reverse turns..
Alpha Structure
Figure: Right Handed Alpha helices -
These helices are formed when the carbonyl O of the i th amino acid H bonds to the amide H of the i th +4 aa (4 amino acids down). The phi/psi angles for those amino acids in the alpha helix are - 57,-47, which emphasizes the regular repeating nature of the structure. It can also be characterized by n (the number of peptide units/turn = 3.6) and pitch (the helix rise/turn = 5.4 angstroms). Some facts:
Chime:
An
isolated helix from an Antifreeze Protein Jmol: An
isolated helix from an Antifreeze Protein
Chime:
Alpha Helix - Web Chime:
Dynamics of
an alpha helix
Note: There are other kinds of helices that can occur. These include a 310 helix and a p helix, which are stabilized by H-bonds between the amide NH and carbonyl O of residues (i, i+3) and (i, i+5), respectively. Likewise, they have 3 and 4.3 residues/turn, respectively, and a rise per residue of 6 and 4.7 angstrom, respectively. These structures are much rarer than right handed alpha helices.
| Helix Type | H bond btw ith and ith+X AA, where X = | Residue/turn | Rise (Angstrom)/turn |
| 310 | 3 | 3 | 6 |
| a | 4 | 3.6 | 5.4 |
| p | 5 | 4.3 | 4.7 |
Beta Structure
Figure: Parallel Beta Strands
Figure: Antiparallel Beta Strand
Beta Structure: Parallel and antiparallel beta strands are much more extended than alpha helices (phi/psi of -57,-47) but not as extended as a fully extended polypeptide chain (with phi/psi angles of +/- 180). The beta sheets are not quit so extended (parallel -119, +113 ; antiparallel, -139, +135), and can be envisioned as rippled sheets. They can be visualized by laying thin, pleated strips of paper side by side to make a "pleated sheet" of paper. Each strip of paper can be pictured as a single peptide strand in which the peptide backbone makes a zig-zag along the strip, with the alpha carbons lying at the folds of the pleats. Each single strand of the beta-sheet can be pictured as a twofold helix, i.e. a helix with 2 residues/turn. The arrangement of each successive peptide plane is pleated due to the tetrahedral nature of the alpha C. The H bonds are interstrand, not intrastrand as in the alpha helix.
Figure: Antiparallel pleated beta sheet
Note: Consider a strand as a continuous and contiguous polypeptide backbone propagating in one direction. Hence, using this definition, a helix consist of a single strand, and all the H-bonds are within the strand (or intrastrand). A beta sheet would then consist of multiple strands, since each "strand" is separated from other "strands" by an intervening contiguous stretch of amino acid which bends within the protein in a way which allows the next section of the peptide backbone, the next "strand" to H-bond with the first "strand". But remember, even in this case, all the H-bonds holding the alpha and beta structure together are intramolecular.
In a parallel beta sheet structure, the optimal H bond pattern leads to a less extended structure (phi/psi of -119, +113) than the optimal arrangement of the H bonds in the antiparallel structure (phi/psi of -139, +135). Also the H bonds in the parallel sheet are bent significantly. (i.e. the carbonyl O on one strand is not exactly opposite the amide H on the adjacent strand, as it is in the antiparallel sheet.) Hence antiparallel beta strands are presumably more stable, even though both are abundantly found in nature. Short parallel beta sheets of 4 strands or less are not common, which might reflect their lower stability.
The side chains in the beta sheet are normal to the plane of the sheet, extending out from the plane on alternating sides. Parallel sheets characteristically distribute hydrophobic side chains on both side of the sheet, while antiparallel sheets are usually arranged with all the hydrophobic residues on one side. This requires an alternation of hydrophilic and hydrophobic side chains in the primary sequence. Antiparallel sheets are found in silk with the sheets running parallel to the silk fibers. The following repeat is found in the primary sequence: (Ser-Gly-Ala-Gly)n), with Gly pointing out from one face, and Ser or Ala from the other.
Chime:
Silk
Beta strands have a tendency to twist in the right hand direction. This leads to important consequences in how the beta strands are connected. Parallel strands can from twisted sheets or saddles as well as beta barrels.
Figure: twisted sheets or saddles as well as beta barrels
Chime:
Twisted
beta sheet from arabinose binding protein | Jmol:
Twisted
beta sheet from arabinose binding protein
Chime:
Beta
barrel from triose phosphate isomerase | Jmol:
Beta
barrel from triose phosphate isomerase
Reverse Turns: About 50% of the amino acids in a globular protein are in regular secondary structure (alpha or beta). The remaining amino acids are not less ordered, just less regular. An additional example of secondary structures is reverse turns (or beta-bends or beta turns). Reverse turns often connect successive antiparallel beta strands and are then called beta hairpins.
Figure: Reverse Turns
They are almost always at the surface, and consist of 4 amino acids. There are two types. (I - f2 = -60, y2=-30; f3 = -90, y3 = 0; II - f2 = -60, y2=120; f3 = 90, y3 = 0 ) Residue 2 of both is often Pro. (Why?) Both have an H bond between the carbonyl O of the i th a.a and the amide H of the i th+3 aa (three amino acids down). In the type 2, the O of residue 2 crowds the beta C of residue 3, so aa2 is usually Gly. Why? Those amino acids which destabilize alpha helices are often found in beta sheets, since the side chains project out of the plan which holds the main chain.
Figure: Type I and Type II Reverse Turns
Chime:
Reverse
Turn from intestinal fatty acid binding protein
Chime:
Reverse Turn Trypsin Inhibitor 2 (aa 20-26, Eballium elaterium)
Jmol: Reverse
Turn Trypsin Inhibitor
(Notice the tightness of the reverse turn and the presence of
Pro and Gly.)
Figure: Why do amino acid propensities for secondary structure differ?
3o Structure
The tertiary structure of a protein is its 3D structure. From the crystal structure of thousands of proteins, common features of protein structure is observed: (Recent Data: new values shown in red. based on much crystallographic data; added 9/26/02 based on paper: Pace, C.N. Biochemistry. 40, pg 310 (2001))
This Chime and Jmol models below show the similarities in the formations of a micelle, in which all nonpolars are buried, to that of protein in which most nonpolar side chain are buried and surrounded in a nonpolar environment.
Jmol:
A
protein with a buried nonpolar amino acid
:
Micelle
Common Motifs Found in Proteins
Super-Secondary Structure - Given the number of possible combinations of 1o, 2o, and 3o structures, one might guess that the 3D structure of each protein is quite distinctive. This is true. However, it has been found that similar substructures are found in proteins. For instance, common secondary structures are often grouped together to form a motifs called super-secondary structure (SSS). See some examples below:
Figure: helix-loop-helix (image made with VMD)
Figure: EF Hand
Chime:
helix-loop-helix from the lambda repressor/DNA Jmol:
helix-loop-helix from the lambda repressor/DNA
Chime:
helix-loop-helix
(EF Hand) from calmodulin Jmol: helix-loop-helix
(EF Hand) from calmodulin
Figure: beta-hairpin, or beta-beta (image made with VMD)
Chime:
beta-hairpin
from bovine pancreatic trypsin inhibitor Jmol:
beta-hairpin
from bovine pancreatic trypsin inhibitor
Figure: Greek Key Motiff
Jmol:
Greek Key
Figure: beta-alpha-beta
Chime: beta-helix-beta
motif from triose phosphate isomerase Jmol: beta-helix-beta
motif from TPI
Jmol
Beta Helices: These right-handed parallel helix structures consists of a contiguous polypeptide chain with three parallel beta strands separated by three turns forming a single rung of a larger helical structure which in total might contain as many as nine rungs. The intrastrand H-bonds are between parallel beta strands in separate rungs. These seem to prevalent in pathogens (bacteria, viruses, toxins) proteins that facilitate binding of the pathogen to a host cell.
Table: Beta Helices
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Vibrio cholerae |
cholera
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Helicobacter pylori |
ulcers |
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Plasmodium falciparum |
malaria |
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Chlamyidia trachomatis |
VD |
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Chlamydophilia pneumoniae |
respiratory infection |
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Trypanosoma brucei |
sleeping sickness |
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Borrelia burgdorferi |
Lymes disease |
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Bordetella parapertussis |
whooping cough |
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Bacillus anthracis |
anthrax |
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Neisseria meningitides |
menigitis |
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Legionaella pneumophilia |
Legionaire’s disease |
Chime:
b - helix
from C. Grisham and E. O'Neil
Domains - Domains are the fundamental unit of 3o structure. It can be considered a chain or part of a chain that can independently fold into a stable tertiary structure. Domains are units of structure but can also be units of function. Some proteins can be cleaved at a single peptide bonds to form two fragments. Often, these can fold independently of each other, and sometimes each unit retains an activity that was present in the uncleaved protein. Sometimes binding sites on the proteins are found in the interface between the structural domains. Many proteins seem to share functional and structure domains, suggesting that the DNA of each shared domain might have arisen from duplication of a primordial gene with a particular structure and function.
Evolution has led towards increasing complexity which has required proteins of new structure and function. Increased and different functionalities in proteins have been obtained with additions of domains to base protein. Chothia (2003) has defined domain in an evolutionary sense as "an evolutionary unit whose coding sequence can be duplicated and/or undergo recombination". Proteins range from small with a single domain (typically from 100-250 amino acids) to large with many domains. From recent analyzes of genomes, new protein functionalities appear to arise from addition or exchange of other domains which, according to Chothia, result from
Structural analyses show that about half of all protein coding sequences in genomes are homologous to other known protein structures. There appears to be about 750 different families of domains (i.e small proteins derived from a common ancestor) in vertebrates, each with about 50 homologous structures. About 430 of these domain families are found in all the genomes that have been solved.
Structual Clases of Proteins - Proteins can be divided into 3 classes of protein, depending on their characteristic secondary structure. Click below for Chime structures showing examples of these proteins.
Chime: cytochrome
B562 Jmol:
cytochrome
B562
Chime:
met-myoglobin Jmol:
met-myoglobin
Chime:
triose
phosphate isomerase Jmol:
triose
phosphate isomerase
Chime:
alcohol dehydrogenase
(with a nucleotide binding domains)
Chime:
hexokinase Jmol:
hexokinase
Chime:
superoxide
dismutase Jmol:
superoxide
dismutase
Chime:
human IgG1 antibody Jmol:
human IgG1 antibody
Chime: immunoglobulin
structure
Chime:
retinol
binding protein Jmol: retinol
binding protein
fatty acid binding proteins; Peptide-N(4)-(N-Acetyl-b-D-Glucosaminyl) Asparagine Amidase (PNGase F) - under construction.
Quarternary Structure
Primary structure is the linear sequence of the protein. Secondary structure is the repetitive structure formed from H-bonds among backbone amide H and carbonyl O atoms. Tertiary structure is the overall 3D structure of the protein. Quaternary structure is the overall structure that arises when tertiary structures aggregate to self to form homodimers, homotrimers, or homopolymers OR aggregate with different proteins to form heteropolymers.
Quaternary Structure from ExPAYs
Globular versus fibril structures
We will deal exclusively with proteins which have a "globular" tertiary structure in this course. However, there are many proteins that form elongated fibrils with properties like elasticity, which measures the extent of deformation with a given force and subsequent return to the original state. Elastic molecules must store energy (go to a higher energy state) when the elongating force is applied, and the energy must be released on return to the equilibrium resting structure. Structures that can store energy and release it when subjected to a force have resiliency. Proteins that stretch with an applied forces include elastin (in blood vessels, lungs and skins where elasticity is required), resilin in insects (which stretches on wing beating), silk, found in spider web) and fibrillin found in most connective tissues and cartilage. Some proteins have high resiliency (90% in elastin and resilin), while others are only partially resilient (35% in silk, which have a tensile strength approaching that of stainless steel. In contrast to rubber, which has an amorphous structure which imparts elasticity, these proteins, although they have a dissimilar amino acid sequence, seem to have a common structure inferred from their DNA sequences. In some (like fibrillin), the protein has a folded b-sheet domain which unfold like a stretched accordion. Others (like elastin and spider silk) have b-sheet domain and other secondary structures (a-helices and (b turns) along with Pro and Ala repetitions. Researcher are studying these structures to help in the synthesis of new elastic and resilient products
SCOP: Structural Characerization of Proteins - Database showing folds, superfamiles, families, and domains
Online Preclass Question
Moodle
Online Quiz (PASSWORD PROTECTED):
PROTEIN 2
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