1. Amino Acids
2. Protein shape is determined mostly by the amino acid sequence.
- Amino acids are hydrophobic, hydrophilic, or have special properties
- Amino acids consist exclusively of the
Proline is actually an "imino acid". Normally there is free rotation about the C-N bond in amino acids. In proline and hydroxyproline, this free rotation is prevented.
- Cysteine can form covalent bonds to another cystine.
3. Proteins tend to fold into the lowest energy conformation.
- Amino acids are covalently bonded from the carboxyl carbon of one amino acid to the amino nitrogen of another amino acid. (fig. 3-3a, Lodish) (fig. 3-3b, Lodish) (fig. 3-3c, Lodish) The planar arrangement of of 6 atoms in the result of resonance. Look
here to see how the resulting protein chain looks like a series of planes, each containing the 6 atoms, linked together.
at this link to see how the planar units of the chain can rotate about the two main-chain bonds of the alpha carbon - the two angles of rotation are phi and psi.
- Only a limited number of combinations of the angles phi and psi occur in nature. These combinations of phi and psi are shown on a
- Proteins consist of a polypeptide backbone with attached side chains.
- The three dimension folding of proteins is the result of specific interactions between side chains. These side chain interactions cause the angles phi and psi to be different for different amino acid sequences.
- Side chain interactions are the result of weak chemical bonds -("Ionic bonds, hydrogen bonds and van der Waals attraction")
- Globular proteins fold with most hydrophobic side chains on the inside (red) and most hydrophilic side chains on the outside (green). This can be summarized in the oil drop model (fig. 3-7, Lodish). But, globular proteins inserted in membranes
often have a band of hydrophobic side chains on the outside of the molecule. This equatorial band fits in the hydrophobic interior of the cell membrane.
- Hydrogen bonds help stabilize the three-dimensional structure of many types of globular and structural proteins.
("Hydrogen bonds in a protein molecule").
- The famous experiment of Anfinsen, Moore, and Stein, which won them a Nobel prize - RNAase, an enzyme (protein of 124 amino acids) was denatured (unfolded) with Urea, which disrupts hydrogen bonds. RNAase also has disulfide bonds, which had to be broken by using a powerful reducing agent, mercaptoethanol, to break the four -S-S-
disulfide bonds from Cystine into -SH HS-. The RNAase became inactive as an enzyme and didn't work anymore. Then the Urea was removed by dialysis and the disulfide bonds reformed by oxidation by oxygen in the air. The enzyme worked once again, indicating that it had refolded into the original three-dimensional structure.
- There may be many low energy configurations - It can be very important for large proteins which end is manufactured first since the chain begins folding even while still attached to the ribosome and while additional chain is being made.
Molecular chaperones and chaparonins are proteins which help other proteins fold and to maintain the folded configuration. Chaparons were first discovered as heat shock proteins, which are produced in response to stress.
- Heat shock proteins were first discovered in 1962 by Ferruccio Ritossa. He showed that stress, like heat, puromycin (an antibiotic), ethanol, heavy metal ions, arsenic, tissue explantation (cutting out some tissue), and viral infection all induced synthesis of certain proteins - HSP70, HSP60, and GRP78 (Glucose-regulated protein). Most of these were shown to form hydrophobic bonds with proteins and help move the hydrophobic regions to the inside when hydrogen bonds had been disrupted by stress (fig. 3-16, Lodish). In normal cells, protection against stress seemed to be the function. But, much later, it was discovered that these proteins also have a function in normal protein synthesis - Many proteins are helped to fold as they are being manufactured or soon thereafter.
- Molecular chaperones and chaparonins are described in Lodish, pp 75-77. Look at (fig. 3-17, Lodish) to see how GroEL, a chaperonin, assists proteins in the folding process.
4. The alpha helix - a common folding pattern.
5. The beta pleated sheet - another common structural feature of proteins.
- Linus Pauling, the Nobel prize winner in Chemistry (and later Peace) predicted the alpha helical pattern from theory, assuming 100% hydrogen bonding between amino and carboxyl groups on the main chain, no blocking of folding by bonding or repulsion between side chains, and known molecular dimensions. He predicted the shape and size of the helix exactly as shown later by experimental studies on Keratin, a protein which is almost pure alpha-helix.
- The alpha helix has 3.6 amino acids/turn, hydrogen bonds which run almost exactly parallel to the helix axis, and 100% hydrogen bonding between main chain groups. (fig. 3-4, Lodish)
- The alpha helix has
overlapping hydrogen bonds. As a result,some can be broken and the structure remains stable. Only when most all of them are broken, does the denatured protein unwind into a random coil configuration.
- Hair consists of almost pure alpha-helix as the protein keratin. The alpha-helical strands which make up a hair shaft are cross-linked with disulfide bonds. (A few of the amino acids are cystine, most are simple amino acids like glycine or alanine, without large or interacting side chains. Permanent waves and hair straightening chemicals are reducing agents which break disulfide bonds. Permanent waves are made by rolling up the hair in curls and letting it reform disulfide bonds, making a "permanent" (until the hair grows out) curl. Hair straightening works the same way except that the hair is kept straight till it "sets".
- Many globular proteins have regions of alpha helix as elements of a larger and more complicated structure
("Regions of alpha-helix"). Look here to see the newly described
chloride channel, which contains many helicies.
- A paper was published in Nature in 2006 that described how both sodium and potassium channels seem to be variations on the same theme. This article, "Membrane biology: Permutations of permeability", is required reading.
- In another recent development, part of the mechanism behind BSE (mad cow disease) has been described. In this disease, a cell membrane protein, PrP, is converted from a form which consists mainly of alpha helicies, is converted to the abnormal form, PrPSc, which has significant beta pleated sheet structure. Look at this figure to see one step in this process of conversion. PrP is shown on the left of the figure and PrPSc is on the right.
6. The collagen helix - an unusual way to make a helical, structural, protein.
- An example of almost pure beta pleated sheet is the protein which makes up silk. (fig. 3-5a, Lodish) (fig. 3-5b, Lodish) Hairpin turns, where the chain reverses direction, are constructed in particular ways- glycine and proline are commonly used. Why are glycine or proline used? What else characterizes the beta turns? (Read Lodish to find out!)
- While silk (produced by the larva of the silkworm moth, to make cocoons) is almost pure antiparallel beta pleated sheet, elements of beta pleated sheet are found in many protein domains.
6. Hemoglobin is an example of a protein with multiple subunits.
- The collagen helix has an unusual amino acid sequence. Approximately every third amino acid is a glycine and most collagens contain an unusually high percentage of proline compared with other proteins. The prolines place kinks in the chain and the entire helix is kept in the helical configuration mainly by these fixed covalent links, NOT the hydrogen bonds as in the alpha helix. The hydrogen bonds are directed outward and help bond adjacent strands of collagen together to form the tropocollagen molecule. The collagen helix has 3.3 amino acids per turn instead of 3.6 as in the alpha helix.(fig. 19-22, Lodish)
- Hemoglobin consists of four nearly identical subunits, each containing a non protein porphyrin ring with an iron atom in the center. The four subunits consist of two types, the alpha and beta subunits. Each of these subunits is very similar to the oxygen storage molecule in muscle, Myoglobin. Here is a small picture of Hemoglobin. Figure 3-5a, Lodish , shows some relationships between different plant and animal hemoglobins.
- Four prolines terminate alpha-helical segments but the other 4 terminations of helical segments probably result from side chain interactions.
- Oxygen binds to the Iron. This causes the Iron atom to move closer to the plane of the porphyrin ring. The pulls a histidine closer to heme plane -> moves the alpha helical segment containing the histidine towards an adjacent helix. This movement pushes a tyrosine which moves the C-terminal (the end of the protein chain with the COO-). In hemoglobin, the C-terminal forms an ionic bond with a positively charged residual group on an adjacent subunit, holding the subunits together. Movement of the C-terminal therefore affects the shape of the adjacent subunit and its ability to bind oxygen.
- Binding of oxygen to hemoglobin is cooperative. Binding of an oxygen molecule to one of the four subunits results in shape changes in the other three subunits, making them more able to bind oxygen. Subsequent binding of oxygen makes the remaining units bind oxygen even more strongly.
- Sickle cell hemoglobin is a genetically based disease in which one nucleotide base is different from normal. This mutation is on the gene which makes the beta chain of hemoglobin and causes a change a different amino acid to be substituted at one location on the beta chain. This substitution causes hemoglobin molecules to bind together in clumps, resulting in water loss from red blood cells. Shrinkage of red blood cells causes them to clog capillaries and prevent normal circulation.
- The gene for sickle cell hemoglobin has a single nucleotide base mutation that causes a substitution of valine (GTG codon) for a glutamic acid (GAG codon) on the beta chain. The sickle cell trait is found in about 8% of African-Americans. Hemoglobin C, found in about 2% of African-Americans, is produced by a gene that has a point mutation at the very same site as for sickle cell hemoglobin, but in this case causing substitution of lysine (AAG codon) for glutamic acid. Hemoglobin C doesn't pose significant health problems for those who have the trait.
- Let's look at some physiological consequences of oxygen binding to myoglobin and hemoglobin -
Myoglobin and Hemoglobin binding curves explained!.
All text and images, not attributed to others, including course examinations and sample questions, are Copyright, 2008, Thomas J. Herbert and may not be used for any commercial purpose without the express written permission of Thomas J. Herbert.