Essential Cell Biology 5th edition

168
PANEL 4–6 PROTEIN STRUCTURE DETERMINATION
X-RAY CRYSTALLOGRAPHY
To determine a protein’s three-dimensional structure—and assess how this conformation changes as the protein
functions—one must be able to “see” the relative positions of the protein’s individual atoms. Since the 1930s, x-ray
crystallography has been the gold standard for the determination of protein structure. This method uses x rays—which have a
wavelength approximately equal to the diameter of a hydrogen atom—to probe the structure of proteins at an atomic level.
To begin, the purified protein is first coaxed into forming crystals: large, highly ordered arrays in which every protein
molecule has the same conformation and is perfectly aligned with its neighbors. The process can take years of trial and error
to find the right conditions to produce high-quality protein crystals. When a narrow beam of x-rays is directed at this crystal,
the atoms in the protein molecules scatter the incoming x-rays. These scattered waves either reinforce or cancel one another,
producing a complex diffraction pattern that is collected by electronic detectors. The position and intensity of each spot in
the x-ray diffraction pattern contain information about the position of the atoms in the protein crystal.
(B)
protein crystal
diffracted beams
beam
stop
x-ray diffraction pattern
obtained from the protein crystal
x-ray source
(A)
beam
of x-rays
calculation of
structure from
diffraction pattern
(C)
(D)
Computers then transform these patterns into maps of the relative spatial positions of the atoms. By combining this information
with the amino acid sequence of the protein, an atomic model of the protein’s structure can be generated. The protein shown
here is ribulose bisphosphate carboxylase (Rubisco), an enzyme that plays a central role in CO 2 fixation during photosynthesis
(discussed in Chapter 14). The protein illustrated is approximately 450 amino acids in length. Nitrogen atoms are shown in blue,
oxygen in red, phosphorus in yellow; and carbon in gray. (B, courtesy of C. Branden; C, courtesy of J. Hajdu and I. Andersson.)
NMR SPECTROSCOPY
If a protein is small—50,000 daltons or less—its
structure in solution can be determined by nuclear
magnetic resonance (NMR) spectroscopy. This
method takes advantage of the fact that for many
atoms—hydrogen in particular—the nucleus is
intrinsically magnetic.
(A)
(Courtesy of P. Kraulis, Uppsala)
(B)
When a solution of pure protein is exposed to a
powerful magnet, its nuclei will act like tiny bar
magnets and align themselves with the magnetic
field. If the protein solution is then bombarded with
a blast of radio waves, the excited nuclei will wobble
around their magnetic axes, and, as they relax back
into the aligned position, they give off a signal that
can be used to reveal their relative positions.
Again, combined with an amino acid sequence, an NMR spectrum can allow the computation of a protein’s three-dimensional
structure. Proteins larger than 50,000 daltons can be broken up into their constituent functional domains before analysis by NMR
spectroscopy. In (A), a two-dimensional NMR spectrum derived from the C-terminal binding domain of the enzyme cellulase is
shown. The spots represent interactions between neighboring H atoms. The structures that satisfy the distance constraints
presented by the NMR spectrum are shown superimposed in (B). This domain, which binds to cellulose, is 36 amino acids in length.