The order of amino acids in a protein molecule is genetically
determined. This primary sequence of amino acids must contain all
the information required for the protein to assume its correct
three-dimensional structure. The primary structure is composed of
amino acids linked together in what are termed peptide bonds. At
first glance these appear to contain only single bonds and free
rotation between all such atoms would be expected.
Measurements of bond angles and lengths of peptide bonds suggests however, that the C = O bond of the carbonyl group is longer than would be expected for such a bond and that the C - N bond is shorter than would be expected. This has led to the concept of the hybrid bonding situation as depicted in Figure 1:
The dotted lines represent the sharing of the electrons that normally would be expected to be found in the double bond. The result is that neither bond behaves as though it were a single or a double bond but rather has intermediate properties. One of the properties it obtains is the lack of rotation between the bonded atoms. This results in the situation as shown in Figure 2:
In this structure there is no rotation about the bond:
This results in the formation of a series of planes as
indicated by the dotted lines in Figure 2.
The alpha 1 carbon, the carbonyl oxygen, the amide hydrogen and the alpha 2 carbon are all arranged in a single plane. Another similar plane is formed with the alpha a 2 and alpha 3 carbons.
The C-C and C-N bonds of alpha 2 C can rotate freely. . As rotation occur at these points, the two adjacent planes will move as intact units. Not all angles between adjacent planes are energetically allowed. Angles that force bulky side chains to come into close contact or for groups with like charges to approach are not allowed.
Ramachandron utilized space filling models and computer simulation to calculate the energies of various combinations of planer angles. When such calculations were made, it was found that most angles yielded areas of high energy and were not allowed. There were, however, areas that yielded minimal values and these angles were the ones that would be expected to be found in protein molecules.
Data obtained from x-ray diffraction of proteins indicates that the predicted angles agreed very well with those observed. Only the amino acid glycine , which contains a hydrogen as an R group, showed major deviation from the predicted angles.
These allowed-angles at areas of low energy correspond to structures that have been observed repeatedly in proteins. This next level of order has been termed secondary structure.
In the early 1950's Pauling and Corey examined the bond lengths and angles of the atoms in amino acids in crystals. They then attempted to devise a repeating structure that would maintain these lengths and also maximize hydrogen bonding. The structure they suggested was a helix that contained 3.6 amino acid residues per turn.
Each peptide can be considered as a plane that is tangent to the axis of the helix. There are 100 degrees of rotation from one amino acid to the next which results in a helix with 3.6 amino acids per turn. Each peptide group; is hydrogen bonded to the third peptide along the chain in either direction. This is a very compact structure that does not have room for water or other small molecules in the interior. A protein that was comprised of only alpha- helix would be a rigid, long, narrow rod. Proteins vary greatly in a helix content from a low of less than 5% to a high of greater than 80%. The first protein to be examined by high resolution x-ray diffraction was myoglobin. About 80% of the amino acid residues in myoglobin were found in one of its 8 helical segments. Most proteins do not contain this much helical structure.
While there is no room in the interior of the helix for even small molecules, interactions of amino acid side chains at its surface are quite common. The residues comprising an alpha helix may be either hydrophobic or hydrophilic and the location of the helix will dictate which type of residue is found. Often a helix is located at the surface of a protein with one side exposed to the solvent and the other side making contact with other portions of the protein molecule. This results in a situation where all hydrophobic groups are found on one side of a helix with only hydrophilic groups on the other side.
The structure of an alpha helix maximizes interactions between amino acid residues. The helical structure also requires that there be favorable interactions between at least three consecutive amino acid residues. this means that a helix doesn't begin to form easily, but once it is formed it is a fairly stable structure. The hydrogen bonds between atoms within the helix are generally not exposed to full contact with the solvent. This lack of solvent interaction causes these hydrogen bonds to be more stable than those observed in small compounds that are more fully hydrated.
To help visualize a helix, the following show a segment of a protein composed of two sections of alpha helix. The first view is a ball and stick representation of the amino acids. The dotted lines indicate hydrogen bonds. The helix is difficult to discern from this representation. The next view is a wire frame representation of the same portion of the molecule. With the amino acids represented in less space, the helical structures are easier to see. The final view is a cartoon representation of the helical structure of the same protein fragment. It may help you to alternate the views and attempt to become better at visualizing the helix.
If a chain of amino acids is drawn in a linear extended conformation, the R groups will fall alternately above and below the plain of the peptide bonds. If another chain of extended amino acids is brought near the first chain, it is a simple matter to line up the chain so as to maximize hydrogen bonding. This can be done whether the chains have the same N to C sense (parallel) or not (anti parallel).
Pauling and Corey first proposed the proper hydrogen bonding scheme for parallel and anti parallel beta structure. The hydrogen bonds are evenly spaced in parallel sheets, but show a more irregular spacing in anti parallel sheets. In both cases, the number of hydrogen bonds formed is maximal. The number of strands involved in beta structure can range from 2 to more than 10. anti parallel sheets are the only ones generally involving only two strands, while both parallel and anti-parallel strands of five or more chains are quite common. Some sheets are mixed containing strands that are bonded to a parallel strand on one side and an anti parallel one on the other. The strands making up the beta structure may be derived from a single chain with no intervening structure or they may be separated by areas of helical structure. In some cases the beta structure may involve amino acids from two or more separate polypeptide chains.
While the R groups of adjacent amino acids appear on opposite sides of the chain, the groups of the separate chains that comprise beta structure are in close proximity. The types of forces described in Chapter 2 are often involved in interaction between these R groups and add stability to the B structures. These forces are stronger in portions of the strands that have been removed from intimate contact with water.
The R groups on one side of a chain tend to be either all hydrophilic or all hydrophobic in anti parallel sheets. Thus, one side of the strand will either favor exposure to the solvent or to other chains that contain hydrophobic regions. In parallel sheets, the R groups tend to be more uniformly hydrophobic and these strands are only rarely exposed to the solvent. Similar to the structures for alpha-helix, the following show beta-structure in either a ball and stick, wire frame and cartoon form.
Large numbers of anti parallel sheets may associate into structures described as barrels. These structures are composed of from 5 to 13 separate strands. The core regions of these barrels are very hydrophobic. Such structures have been identified in a number of proteins and have been reported to have about the same cross sectional area regardless of the number of strands they are composed of. The conditions required for the formation of beta structure are not as rigid as for the formation of alpha helix. In many cases a couple of amino acids having an extended structure can be hydrogen bonded to another couple of amino acids that are separated by a large number of intervening amino acids. Such interactions should be considered as beta structure. The following show beta barrels in either a ball and stick, wire frame and cartoon form.
When a chain folds back on itself to form an anti parallel beta sheet, the conformation of the amino acids in the turn portion are generally in one of two conformations. (Figure 3). The structures shown are the classic beta bends. Many variations of these have been found and some authors suggest there are up to ten types of turns with variations for each type.
The original beta bends. were recognized to be stabilized by hydrogen bonding within the four amino acids composing the bends. As similar structures have been located in additional proteins, it has been observed that almost half of them are stable without the occurrence of any hydrogen bonding. In these cases, just the normal energy constraints of the bond angles lead to stable structures.
Beta bends. are important to protein structure. These small structures direct the main chains of proteins into directions that allow for proper interactions. Depending on the criteria for defining beta bends, they have been estimated to consist of from 20 to 45% of the total structure of all proteins.
At one time it was popular to refer to all secondary structure of proteins that was not alpha-helix or beta sheet as random coil. This term gives the unfortunate connotation of a random, dynamic structure for much of the protein. What is really meant, is that no readily apparent repeating structure is present. The fact that proteins form well-defined crystals and that sharp x-ray diffraction patterns can be obtained form these crystals, suggests that essentially all of the molecules of a given type of protein are in the same three-dimensional conformation. Thus random should, in this case, be used to suggest no readily apparent repeating structure rather than a truly random location of the amino acid residues.
Beta bends. were the first non repeating, yet organized structures that have been recognized in proteins. Their greatest contribution to the structure of proteins may be their direction of protein folding. It remains to be seen if other regular non-repeating structures will be recognized in proteins.
The following show a turn in either a ball and stick, wire frame and cartoon form.
The final three dimensional structure a protein assumes is called its tertiary structure. This level of structure defines the location of each amino acid of the protein in three-dimensional space. Tertiary structure may be considered as being the same as the conformation of the protein. With few exceptions proteins are not long extended structures, but have dimensions that are not too different from spheres or ovoids. This suggest that once secondary structures have formed the molecules fold into relatively compact structures.
The specific tertiary structure assumed by a protein can have considerable impact on the properties of the molecule. The protein folds in such a way as to remove as many hydrophobic groups as is possible from contact with the aqueous phase. The final conformation should also attempt to maximize favorable interactions between different portions of the molecule. This usually results in a molecule having a very compact interior. The hydrophobic groups are associated away from the water and are able to interact due to London forces. the interior of the molecule is usually devoid of water molecules or of charged amino acid residues. The energy required to over come the interactions of charged groups with water that would be necessary for their insertion into the protein interior is generally not available. When a protein does find the necessary energy to bury a charged group or even a dipole,the buried group usually can be shown to perform some specific function necessary to the functionality of the protein
While the protein maximizes interactions between its constituent amino acids it will also optimize the number of interactions with the solvent. To a large extent the number and strength of these interactions will determine the solubility characteristics of the protein. It should be noted that the specific nature of the interactions that occur and thus the conformation of the protein are greatly dependent upon the environment. Changes in temperature, ionic strength, dielectric constant, pH, etc. would be expected to have affects on the structure of the protein. These changes may be very subtle or of great consequence to the structure and function of the molecule. Whenever the conformation of a protein is discussed the conditions must be specified so that others will be able to reproduce the observations.
The structures previously described as beta sheets are technically tertiary structures. the secondary structure is the extended chain of amino acids. The interaction of two or more chains that leads to the formation of beta structures are more properly classified as tertiary. Most workers include discussions of beta sheets with considerations of secondary structures. As long as it is clear that the extended structure is the real secondary structure there is no problem with this type of treatment.
The small protein lysozyme, molecular weight about 14,000, can be used to help visualize the tertiary structure of proteins. The first view of lysozyme is a space filling view. No evidence of secondary or tertiary structure is evident from this view. Removal of the depth of the atoms gievs the wire frame view. Some hydrogen bonds can no be seen as dots. The color scheme for this representation has changed. Alpha helical structures appear as megenta, beta structures are yellow, turns are light blue and all other structures are white. This color scheme will be used in all subsequent representations of the lysozyme molecule. The color clues make visulization of the secondary features a little easier. We can also see how the secondary structes are arranged into tertiary structure. A back bone structure makes the visualization even easier. The hydrogen bonds are much clearer and the tertiary structure of the protein is evident. The final view is a cartoon representation of the tertiary structure.
A much larger molecule carboxypeptidase A, molecular weight about 94,000, is shown in the ball and stick, back bone and cartoon representations. As the complexity of the molecule increases, visualization of the tertiary strcuture becomes more difficult. As the figures indicate, the number of tertiary interactions also increase with size and complexity.
Many protein molecules tend to associate in well-defined structures. Such associations are termed quaternary structures. These structures are often caused by the addition of small molecules or by slight changes in the structure of the individual molecules. Many enzymes, for instance, can be polymerized or depolymerized by the action of phosphatases or kinases. The addition or removal of a phosphate group from a protein molecule can greatly change its tendency to form associated structures.
In many cases an organism can rapidly change the activity of an enzyme by such modification of its quaternary structure. These modifications can be accomplished very rapidly and are readily reversible. This allows for rapid control of enzyme activity.
Many proteins of importance in food systems exhibit quaternary structure. Soy globulins, casein and actomyosin are just a few examples of such proteins. When these proteins are discussed in later chapters, the importance of quaternary structure to their functional properties will be discussed.
The enzyme, Lactate Dehydrogenase, provides an example of quaternary structure. Four chains come together to form the complex shown in the back bone and cartoon form. In the following cartoon, the color scheme has been changed so that each separate chain is a different color. Many foods contain far more complex quarternary structures that will be discussed at the appropraite time.