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4 Tertiary Structure: Proteins Exhibit Common Folds
3.4 Tertiary Structure: Proteins Exhibit Common Folds
Fig. 3.9 Some common protein folds
Fig. 3.10 (a) The structure of a globin reveals many polar groups (blue and red) on the surface.
(b) If the surface is removed to reveal the inside of the protein, (c) there are predominately amino
acids with hydrophobic side chains (grey) present
individual domains that are tethered together by short stretches of peptide. Domain
structures are thought to result from the combination of gene products that work
together toward a common goal into single coding regions.
An example of an enzyme with discrete domains is flavohemoglobin, which
detoxifies nitric oxide (Fig. 3.11). This enzyme uses the “nitric oxide deoxygenase”
reaction to convert nitric oxide to nitrate. This reaction requires oxygen binding,
followed by reaction of nitric oxide with the oxygenated complex. The resulting
ferric hemoglobin must be reduced to the ferrous state to continue the reaction.
Flavohemoglobin has evolved a hemoglobin domain to bind oxygen and react with
nitric oxide, and a flavoprotein domain to reduce the hemoglobin.
3 Protein Structure and Function
Fig. 3.11 Flavohemoglobin,
a protein with two discrete
folding domains. The
hemoglobin domain is
shown in green, and the
flavoprotein in red.
The dashed line shows
the interface between the
Quaternary structure results from the combination of individual polypeptide chains
into larger proteins with multiple subunits. The complexity of quaternary structure
can range from homodimeric proteins, to large complexes making up viral coats,
to megadalton combinations of protein and nucleic acid that constitute organelles
like the ribosome. The forces that hold subunits together are the same that drive
protein folding in the first place: multiple weak forces and the hydrophobic effect.
Most proteins with quaternary structure are held together by buried hydrophobic
surface at the subunit interfaces in combination with precisely placed electrostatic
interactions. The special arrangement of hydrophobic and electrostatic regions on
the subunit surface complement each other like pieces of a puzzle.
Sometimes quaternary structure is stabilized through disulfide bonds between
subunits. This is relatively common in extracellular proteins which must remain
intact outside of the protein-concentrated, controlled environment of the cell.
Classic examples of these molecules include insulin and other peptide hormones,
and many proteolytic enzymes.
What Are Protein Structures and How Are Protein
All of the images we have seen in this chapter are artist’s renditions of the structures
of proteins. Are alpha helices and beta sheets really flat ribbons? Clearly not, but
these representations are useful for helping students learn the important elements of
the structures. All images of protein structures start as files containing the threedimensional coordinates of every atom in the molecule. These coordinates are
measured experimentally, and stored for use by scientists in the “Protein Data
Bank,” along with the file designation “pdb” (Fig. 3.12). These atomic coordinates
can then be plotted and rendered artistically in many different ways, leading to the
3.6 What Are Protein Structures and How Are Protein Structures Measured?
Fig. 3.12 Protein structures are artist’s renditions of the molecular coordinates from a “pdb” file
wealth of molecular images that adorn the covers and pages of biology and
The first atomic resolution protein structure to be measured was that of myoglobin
from sperm whale, in 1957. John Kendrew and Max Perutz received the 1962 Nobel
Prize in chemistry for this contribution, and that of the first structure of red blood cell
hemoglobin published in 1960. The technique of X-ray crystallography was used to
measure these structures, and is still the technique most often used to solve new
structures today. Two big problems that faced the first structural biologists were how
to resolve objects as close together as organic chemical bonds (on the order of 1.5 A
and how to prevent rotational averaging of the molecule during data collection.
The first problem concerns the fact that radiation used to resolve objects cannot
be much longer in wavelength than the size of the objects to be resolved. For
example, our eyes use visible light (~500 nm in wavelength) to see objects down to
the size of ~1/10 of a millimeter. At that point, our retinas become limiting, but if
we use a light microscope for magnification, we can see objects down to ~1 mM
(approximately twice the wavelength of visible light). At that point, however,
visible light can no longer resolve smaller objects. To resolve objects on the
˚ , we need radiation with a wavelength near or shorter than this.
order of 1.5 A
X-rays, which are easy to produce and relatively inert, meet this need.
When molecules are present in solution, they exist in all manners of rotation and
translation throughout the container. If such molecules were exposed to X-rays, the
image taken would reveal rotationally averaged, lacking high-resolution information about the relative locations of each atom. To achieve this level of imaging, the
molecules must be fixed into one or a small number of orientations. This requirement is met by the use of single crystals for X-ray diffraction studies. Molecules in
the crystalline form are fixed in place in a regular, repeating fashion, facilitating
atomic resolution imaging.
In the X-ray diffraction experiment, patterns of scattered X-rays from the protein
crystal are compared to calculations of those that would result from potential
structures inside the same crystal lattice. Good correlation between predicted and
3 Protein Structure and Function
observed scattering indicates an accurate structure. Once a source of highconcentration (usually millimolar), pure sample is available, the limit to the ability
to solve macromolecular structures by X-ray crystallography is growing diffraction
quality crystals. There are no limits brought by molecular size or complexity.
The other technique suitable for measuring atomic resolution structures of
macromolecules is nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy takes advantage of the fact that the unique molecular environment of
each nucleus can be measured, and leaves a measurable fingerprint on neighboring
nuclei. NMR experiments can measure these interactions to provide a collection of
distance restraints that exist among the nuclei in the molecule. The process of
solving the structure using NMR involves computationally manipulating the collection of bonded atoms into different conformations until one matching the
observed contacts is found. Advantages of NMR for molecular structure solution
include the ability to work in solution rather than the crystalline state, but there
is a limit of approximately 50 kDa to the molecular size that can be studied at
Hemoglobin: An Example of Protein Structure
Prior to the development of recombinant genetic techniques, biochemists (and
particularly structural biologists) were left to study proteins that were naturally
available at relatively high concentrations. These included molecules that one
could “grind and find,” meaning that purification must have started from natural
sources and tissues. One of these proteins is red blood cell hemoglobin (Fig. 3.13).
Physiological studies had previously revealed that the function of hemoglobin is
Fig. 3.13 (a) Red blood cells contain high (>30 mM) concentrations of the protein hemoglobin,
which transports oxygen in mammals. The red color comes from the heme prosthetic group
(b), which contains an iron atom (shown in brown) responsible for most of the color
3.7 Hemoglobin: An Example of Protein Structure and Function
oxygen transport, and the tools used to study protein structure and function were
developed largely with the goal of learning how hemoglobin structure confers this
Unlike most enzymes, hemoglobin does not catalyze chemical reactions as part
of its principal natural function. It simply binds and releases oxygen; it binds
in the lungs where oxygen is plentiful, then releases it to tissues that would not
otherwise have access to oxygen. Thus, while it is not catalyzing net chemical
reactions, it is catalyzing oxygen transport. Under the simplest circumstances, the
reversible binding of oxygen (O2) to a protein (P) can be described by the
P ỵ O2 é PO2 :
The dissociation equilibrium constant for the reaction is:
If we are interested in measuring the fraction of protein bound to oxygen
at different oxygen concentrations, we can define the fractional saturation of protein as:
ẵP ỵ ẵPO2
Equation (3.3) will be zero if no oxygen is bound, and 1 if all of the protein is oxygenated. If we substitute for PO2 in (3.3) with (3.2), we get the
following expression for fractional saturation as a function of oxygen concentration and K.
ẵK ỵ ẵO2
Equation (3.4) tells us that the degree to which our protein is saturated with
oxygen depends on the dissociation equilibrium constant and the oxygen concentration, both of which have units of concentration. A convenient way to think about
(3.4) is to set Y ¼ 0.5. At this value, K must be equation to oxygen concentration.
Thus, K is the oxygen concentration at which P will be half-saturated.
The shape of the binding curve resulting from (3.4) is shown in Fig. 3.14a, using
as an example hemoglobin with a value of K ¼ 26 Torr (that of red blood cell
(Torr is a unit of pressure measured by the level of mercury in a manometer.
It can also be used as a unit of concentration for oxygen, along with the known
value for oxygen solubility, as it reflects the oxygen concentration in solution