PROTEIN STRUCTURE
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:
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:
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.
a-Helix
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 a- 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 a-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 a-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.
Beta-Structure
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 b-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 a-helix, the
following show b-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.
Beta bends
When a chain folds back on itself to form an anti parallel b-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.
