DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 19

Methods to determine secondary and tertiary
protein structure
While MS is now widely used to determine partial sequences of pro-
teins for purposes of proteomic analysis, it does not proved information
about the secondary or three-dimensional structure of protein. Methods
that are commonly employed for these purposes are circular dichro-
ism for secondary structure analysis and x-ray diffraction and nuclear
magnetic resonance (NMR) for obtaining 3-dimenstional structures of
proteins. X-ray diffraction and NMR are frequently considered comple-
mentary techniques. The importance of both x-ray diffraction and NMR
for the structural analysis of proteins cannot be underestimated: the de-
velopment and use of these methods have led to several Nobel Prized
(seeTable1).
Circular dichroism (CD) spectroscopy
Circular dichroism is a method that provides basic information on
the overall secondary structure of a protein, including the percentage
of beta sheets and alpha helices. Random coil structures also gener-
ate characteristic CD spectra. CD spectroscopy measures differences
in the absorption of left-and right-handed polarized light that arises from
asymmetric structures. Secondary structural analyses are usually car-
ried out in the “far-uv” spectrum (190-250 nm). Using CD spectroscopy
in the “near-uv” spectrum (250-350 nm) can sometimes provide some
information on the tertiary structure of the protein. CD spectroscopy
is also used to measure structural changes that might occur upon the
interaction of two proteins, or upon receptor-ligand interactions.
X-ray crystallography
X-ray crystallography was the first method developed for determin-
ing the 3-dimenstional structure of a protein and remains the method of
choice for solving the structure of proteins that can be crystallized. X-ray
crystallography has also been used to elucidate the structure of multi-
protein complexes and protein-DNA complexes. The method is based
on principles established over ninety years ago, and referred to a as
Bragg's Law. The Braggs (a father and son “dynamic duo”), shared the
Nobel Prize in Physics in 1915 for their work demonstrating that x-rays
could be used for structural analysis, a year after Max von Laue re-
ceive the Nobel Prize for demonstrating that crystals diffracted x-rays.
Bragg's law (without going into the physics) allows information about a