Page 155 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Encyclopedia of Physical Science and Technology EN013D-616 July 27, 2001 12:5

Protein Structure 195

modification can only be obtained through chemical or structure prediction. Although considerable progress has
physical experimentation. been made in recent years toward establishment of a com-
prehensive structural database many more protein models
B. Cofactors are needed before structures can be predicted with a high
degree of confidence. There are two methods by which
Although many proteins derive all of their function from protein structures can be determined: X-ray crystallog-
their constitutive amino acids, a large number of pro- raphy and NMR. These techniques are complementary,
teins require additional cofactors in order to fulfil their with each having its advantages for providing informa-
biological role. These cofactors provide chemical prop- tion about specific aspects of protein structure. A detailed
erties that are not present in the 20 amino acid residues. description of these methods is beyond the scope of this
For example, none of the amino acids are capable of fa- summary, but a few comments are noteworthy
cilitating an oxidation/reduction reaction. A wide range
of cofactors are utilized including inorganic ions such as
2+
2+
2+
Fe ,Mg , and Zn , or complex organic molecules that A. X-Ray Crystallography
are normally described as coenzymes such as flavin ade- The first structure of a protein, myoglobin, was determined
nine dinucleotide or nicotinamide adenine dinucleotide. by X-ray crystallography in 1958 and was followed soon
If the coenzyme is covalently bound to the enzyme it thereafter by the structure of hemoglobin. At that time
is often called a prosthetic group. The complete en- protein structure determination was a daunting undertak-
zyme is called a holoenzyme, whereas the protein in ing and few structures were determined in the ensuing
the absence of its cofactors is called an apoenzyme or years. Fortunately continual developments in the funda-
apoprotein. Many apoproteins are considerably less sta- mental understanding of X-ray crystallographic theory,
ble in the absence of their cofactor. This suggests that al- data collection, and computational methods have made
though the amino acid sequence dictates the overall three-
the determination of protein structure routine. The result
dimensional structure, the cofactor is an integral part of the of this approach is an electron density map, which is in-
protein. terpreted in terms of a molecular model. The strength of
this technique is that it can be applied to any macromolec-
C. Context Determines Function ular assembly that can be crystallized. The overwhelming
majority of structures in the protein databank have been
Typically proteins and especially enzymes contain only a
determined by X-ray crystallography.
few residues that are absolutely vital for function. In con-
The limiting factor in a successful X-ray structure deter-
trast there are usually many other residues of the same
mination is the growth of high quality crystals. In general
type in the protein that do not fulfill any special role. In
if suitable crystals can be obtained a three-dimensional
many cases the catalytic residues have different chemi-
structure will be determined. The final quality of an X-ray
cal and physical properties from the same amino acid in
structure is directly dependent on the three-dimensional
solution; for example, the pK a of the side chain might
order of the crystals since X-ray crystallography is an
be several pH units higher or lower than the free amino
imaging technique. This is usually indicated by the “res-
acid. It is generally found that the behavior of an amino
olution” of the data. Resolution refers to the minimum
acid is profoundly influenced by the context of that amino
diffraction spacing included in the structural determina-
acid within the protein. Altering the chemical properties
tion where a smaller the number corresponds to a better
of a functional group is one of the major attributes of pro-
˚
structure. Typically a structure at 2.8 A resolution is satis-
tein structure and appears to be essential for the activity
factory to determine the path of the polypeptide chain, but
of most enzymes. There are many ways in which this is
˚
data better than 2.5 A are required to define the hydrogen
achieved; however, a simple example is placement of a
bonding pattern in a protein with great confidence.
charged residue in the interior of a protein such that the
The one concern leveled at X-ray structures is the influ-
deionized state is favored. This serves to raise the pK a of
ence of the crystalline lattice on the observed conforma-
aspartate and glutamate and lower the pK a of lysine.
tion of the protein. Fortunately it has been demonstrated
repeatedly that the structures of proteins observed in
III. PROTEIN STRUCTURE crystallinelatticeareconsistentwithmostofthebiochemi-
DETERMINATION cal measurements on the same protein. This arises because
protein crystals typically contain about 50% solvent such
A major requirement for understanding protein structure that very little of a protein molecule is in contact with
is a large database of three-dimensional structures. This its neighbors in the crystal lattice and the packing forces
is particularly important for the comparative method of are thermodynamically small. In some cases proteins are