Examples of Protein Structure
(Ch 6 - 174-185)
Hemoglobin
(Ch 7 - pgs 214-244)
Characterization of Proteins
(Ch 2 - pgs 50-53), (Ch 5 - pgs 152-163)
Problems
(Ch 2 - 16; Ch 7 - 2,6,8)
Practice Problems
Finish the slide show, practice questions, and text reading, then click below to go to the assignment:
Assignment - Protein Data Base
Protein structures exhibit immense variability. Let us start by investigating the regular repeating patterns found in some fibrous proteins (silk fibroin, keratin, and collagen).
Notice the location of the side chains in the following figure (the red lines indicate oxygen, the white y-shaped lines indicate methyl):
The pleated sheets are fully extended, strong, yet flexible since the sheets are only held together by weak associations, not covalent bonds. Furthermore, silk fibroin is elastic due to "interruptions" in the beta pleated sheet. For example, the regular repeating structure is interrupted by a valine or tyrosine after about 100 amino acids. These amino acids with larger side chains result in less weak bonding between the polypeptide backbone and thus more elasticity in the protein.
In the structure described above, the keratin can be stretched since the alpha helix is NOT an extended structure. When the helix is stretched, the polypeptide chains align and they form a beta-sheet. Since larger side chains do not fit within the beta-sheet structure, the protein returns to the helical microfibril. When the side chains are smaller (such as in hooves and claws), then the beta-sheet is a stable structure and the keratin is much harder.
Keratin is known to have many disulfide bonds. Stronger forms of keratin are known to have more disulfide bonds. The permanent waving of hair is carried out by first reducing all the disulfide bonds to -SH (sulfhydryl groups); then, curling the hair and re-oxidizing to form disulfide bonds in the new positions (-S-S-).
In order to generate the integrity of the final collagen molecule, two important structures are required:
The bonds formed by the lysine cross-links also strengthen the collagen. Over time, as more of these lysine cross-links form, the collagen becomes more rigid in a continuum from very soft infant bones to rigid, more-readily broken bones of the elderly.
The proteins described above are fibrous in nature; however, many proteins are globular in shape. The globular proteins are rarely used for structural applications, but more often have binding or enzymatic roles. Hemoglobin and myoglobin are examples of globular proteins.
Myoglobin (Mb) is a compact, single-subunit protein in which nearly 75% of all amino acids are incorporated into alpha-helices. The interior of the globular protein is nearly exclusively comprised of nonpolar amino acids with the exception of two histidines. The Mb molecule is associated with a porphyrin, heme (see page 217), which is bound via weak associations to the core of the protein. The center of the heme group contains an Fe atom, coordinated to four nitrogens in the porphyrin and two nitrogens from two histidine side chains (see page 218).
The Fe is essential to the function of the heme group as an oxygen carrier. The Fe directly binds to the oxygen molecule--the oxygen displaces one of the histidines coordinating the Fe ion. For this oxygen binding, the Fe must be in the 2+ oxidation state. If the Fe is oxidized to the 3+ oxidation state, the oxygen is unable to bind.
So why do we need such an intricate Mb molecule if only the Fe binds to the oxygen? In the absence of Mb, the Fe-porphyrin complex oxidizes to Fe(III) and dimerizes with another Fe-porphyrin complex. The protein part of the Mb provides the steric constraints that prevent the oxidation of the Fe(II) and the dimerization of the porphyrins.
Hemoglobin (Hb) is a multisubunit protein containing two alpha and two beta subunits that individually appear very similar to Mb, but function quite differently together. The quaternary structure of the Hb allows the Hb to have allosteric regulation. The following figure illustrates the 3D structure of Hb. Notice the four similar subunits and the four independent oxygen binding sites in the centers of the porphyrin rings. Each subunit of the Hb is a different color; each porphyrin ring is shown in red.
Imagine the Hb molecules in the red blood cells, moving between the lungs and the skeletal muscle. The Hb acquires oxygen in the lungs and moves out to the skeletal muscle to deliver that oxygen. What signal indicates to the Hb that the oxygen should be released when the Hb molecule reaches the skeletal muscle cells? The answer lies with the allosteric regulation.
Each subunit of the Hb molecule has an oxygen binding site, but the first subunit to bind oxygen binds with the greatest affinity. After this first oxygen binds, the protein undergoes a conformational change (the 3D structure changes) and the oxygen affinity for the remaining subunits subsequently changes. See the oxygen binding curves on page 221 of the textbook. As the concentration of oxygen increases so does the oxygen saturation of the Hb, but the relationship is NOT linear. The first oxygen binds with the greatest affinity leading to a sigmoidal shaped oxygen binding curve.
Be sure to contrast the Hb and Mb oxygen binding curves. Myoglobin does NOT exhibit allosteric regulation and its oxygen binding curve is much steeper; the first, and only oxygen, binds tightly.
Although these oxygen binding curves explain the binding of oxygen in the lungs and release at lower oxygen concentrations such as in skeletal muscle, they do not explain the physical changes that take place in the Hb.
Think about the following questions related to the Hb molecule (answers are provided after the questions):
Be sure to understand the cause of sickle cell anemia, described in Chapter 7 of the textbook.
Proteins are a very complex, diverse classification of molecules. Proteins differ in size, charge, shape, function, and sequence. One of the few simplifying features of protein structure is that the amino acid sequence must be linear and NOT branched. Furthermore, proteins are difficult to isolate in their pure form. Often the proteins are mixed with numerous proteins that may be similar in structure. Yet, we are often interested in characterizing a particular protein (to investigate its drug potential, to understand a metabolic process better, to isolate the protein for industrial or medical applications).
We will describe several experimental techniques for characterizing and purifying protein molecules (NOTE: The understanding of experimental techniques can be difficult. We will discuss all of these techniques in more detail in the first lecture meeting!):
See pages 50-53 for some introduction. Electrophoresis is based on separating proteins based on their differences in net charge. Depending upon the amino acid sequence and the pH, each protein is expected to have a slightly different net charge. For an oversimplified example consider two proteins at pH 7, one with an abundance of lys and one with an abundance of asp. Each of these proteins will have a very different net charge (many lys lead to a +++++ charge, while many asp lead to a ----- charge).
In the simplest electrophoresis experiments using paper or gels, the proteins are placed in the center of the plate (on the salt-saturated paper or gel); then, an electric field is applied to the plate. The electric field makes one side of the plate more positive (anode) and the opposite side more negative (cathode). Proteins are drawn to the pole that is opposite to its net charge. Ideally, the mixture of proteins is different enough to cause some proteins to migrate further from the center line than others as is shown below:
Proteins a and b have a net negative charge at the pH on the electrophoresis gel; proteins c, d, and e have a net positive charge at the pH of the gel. If the electric field is removed, the proteins diffuse through the gel randomly.
The ability of this simple electrophoresis experiment to separate proteins is limited by the diffusion of the proteins in the gel or on the paper. There is a much higher resolution technique for separating proteins called PAGE (PolyAcrylamide Gel Electrophoresis):
PAGE is based on the premise that all proteins have some hydrophobic residues that will bind to a detergent such as SDS (sodium dodecyl sulfate). As the name suggests, SDS has a long segment of 12 carbons terminating with a negatively charged sulfate group. Somewhat surprisingly, the SDS binds to proteins in similar amounts based on the molecular weight of the protein. The first step in PAGE is to mix the proteins with SDS such that each protein is denatured and is surrounded by a ring of negative charge.
All the proteins appear as spheres of negative charge. The SDS-protein is loaded onto the top of a gel formed from polyacrylamide (a cross-linked polymer with spaces that allow small proteins to pass through while larger proteins are retained). When the electric field is applied the bottom of the gel is the anode and draws all the negatively charged proteins toward it.
Which proteins move the furthest? This is a bit of a trick question. The answer is that the smaller proteins move farther because the larger proteins are retarded by the gel, BUT certainly the smaller proteins also have less negative charge. Experimentally, we have found that the amount of negative charge has little effect on the protein's movement through the gel. Thus, smaller proteins (with less charge) move further through the gel. The following figure shows a completed SDS-PAGE experiment with one protein sample containing three proteins (a,b,c) alongside a mixture of two standard proteins.
Since proteins move according to their size, the position of the protein in the gel indicates the molecule weight of the protein. Protein c is the protein with the lowest molecular weight.
The charge on a protein is determined by the pH of the environment. As the pH increases, more of the amino acid side chains are deprotonated and become negatively charged. As the pH decreases, more of the protons are removed and the protein becomes more positively charged. At some pH, a protein will have a net charge of zero--the positive and negative charges will cancel one another. This pH is known as the isoelectric point (pI).
In isoelectric focussing, a gel is used in which a pH gradient has been established using polyampholytes (lower pH at the top and higher pH at the bottom). When the experiment begins, the protein is at the top of the gel and an electric field is established. Proteins will move through the electric field, attracted to the end of the gel with opposite charge to itself. Proteins become immobile when the charge on the protein is zero (it is not driven to the anode or the cathode). At what pH does the protein have no net charge? The pI for the protein.
In the column shown above, a positively charged protein is repelled from the top of the column; as the protein moves downward to higher pH values, protons are lost. Then, the protein becomes less positively charged until it reaches its isoelectric point. This process only works because the pH gradient and the charge gradient both contribute to the environment around the protein.
See pages 153-155 for some introduction. Since molecular weight and net charge differ between proteins, both of these properties can be used to separate proteins from one another.
For example, we can use a column filled with fixed charges to separate proteins. This technique is known as ion exchange chromatography and was used in week 1 of this course to separate amino acids.
For example, we can use a column filled with porous beads to separate proteins by their different molecular weights. These columns are called gel filtration columns. Large proteins do not fit into the porous beads and take a direct route right through the column. In contrast, small proteins fit into the porous beads and are retarded in their movement through the column.
Protein A is a large protein that moves right through the ion exchange column. Protein B is a smaller protein which is able to enter the beads in the gel filtration column. Since protein B enters the beads, it takes longer for protein B to pass through the column. Using this technique, we can readily separate proteins with differing molecular weights.
Sickle Cell Hemoglobin Animation
