Proteins #4

Determination of Protein Structure

and Protein Engineering
X-Ray Crystallography
• X-rays are a form of electromagnetic
radiation with a wavelength typically
around 0.1nm (the diameter of a
hydrogen atom).

• If a narrow parallel beam of x-rays is

directed at a sample of pure protein, most
of the x-rays pass straight through it.

• A small fraction will however be scattered

by the atoms in the sample. If the sample
is a well ordered crystal, the scattered
waves reinforce one another at certain
points and appear as diffraction spots
when the x-rays are recorded by a
suitable detector.
X-Ray Crystallography
Diamond U.K.
• The position and intensity of each spot
contains information about the locations
of the atoms within the crystal that gave
rise to it.

• Deducing the three dimensional structure

of a large molecule form the diffraction
pattern is a complicated task and was not
achieved for a protein molecule until the
1960s.

• An electron density map is produced

from the overall diffraction pattern using
a mathematical technique called a
Fourier transform.

• A model for the structure is then built

that is consistent with the electron-
density map.
 Problems
• In recent years X-ray diffraction has become increasingly
automated, however many problems still remain with the
technique.

• The slowest step is often the generation of suitable crystals.

• This requires large amounts of very pure protein and often

involves many years of trial and error, searching for the proper
crystallization conditions.

• There are still many proteins, in particular membrane proteins,

which have so far resisted all attempts to crystallize them.
• The physical environment of the crystal is not
identical with that within a living cell.

• A crystal imposes a space and time average on

the structure deduced from the analysis and thus
provides little information about molecular
motion within the protein (e.g. calmodulin).

• The conformation of the molecules within the

crystal can also be affected by nonphsysiological
factors such as incidental protein-protein
interactions.
• Structures derived through this method
should always be confirmed by comparing
structural data acquired by other means.

• Also mobile portions of the protein are

difficult to determine as they may have a
different confirmation in different protein
molecules within the crystal.
 Nuclear Magnetic Resonance (NMR)
Spectroscopy
• This technique depends on the fact that certain atomic nuclei are
intrinsically magnetic.

• The simplest example is the hydrogen nucleus (1H), which is a

proton.

• The spinning of a proton generates a magnetic moment. This

moment can take either of two orientations, or spin states (called α
and β), when an external magnetic field is applied.

• The energy difference between these states is proportional to the

strength of the imposed magnetic field.
• The α state has a slightly lower energy and hence is
slightly more populated (by a factor of the order of
1.00001 in a typical experiment) because it is aligned
with the field.

• A spinning proton in an α state can be raised to an

excited state (β state) by applying a pulse of
electromagnetic radiation (a radio-frequency, or RF,
pulse), provided the frequency corresponds to the
energy difference between the α and the β states.

• In these circumstances, the spin will change from α to β;

in other words, resonance will be obtained.
• A resonance spectrum for a molecule can be
obtained by varying the magnetic field at a
constant frequency of electromagnetic
radiation or by keeping the magnetic field
constant and varying electromagnetic radiation.
• These properties can be used to examine the chemical
surroundings of the hydrogen nucleus.

• The flow of electrons around a magnetic nucleus generates

a small local magnetic field that opposes the applied field.

• The degree of such shielding depends on the surrounding

electron density. Consequently, nuclei in different
environments will change states, or resonate, at slightly
different field strengths or radiation frequencies.

• The nuclei of the perturbed sample absorb

electromagnetic radiation at a frequency that can be
measured.
• The different frequencies, termed chemical shifts, are expressed
in fractional units δ (parts per million, or ppm) relative to the
shifts of a standard compound, such as a water-soluble
derivative of tetramethysilane, that is added with the sample.
• For example, a -CH3 proton typically exhibits a chemical shift (δ)
of 1 ppm, compared with a chemical shift of 7 ppm for an
aromatic proton.
Increasing Complexity
• It is possible to resolve most protons in many
proteins by using this technique of one-
dimensional NMR.

• With this information, we can then deduce

changes to a particular chemical group under
different conditions, such as the
conformational change of a protein from a
disordered structure to an α helix in response
to a change in pH.
 NOESY
• We can garner even more information by
examining how the spins on different protons
affect their neighbors.

• By inducing a transient magnetization in a

sample through the application a radio-
frequency pulse, it is possible to alter the spin
on one nucleus and examine the effect on the
spin of a neighbouring nucleus.
 NOESY
• Especially revealing is a two-dimensional
spectrum obtained by Nuclear Overhauser
Enhancement Spectroscopy (NOESY), which
graphically displays pairs of protons that are in
close proximity, even if they are not close
together in the primary structure
• The basis for this technique is the nuclear Overhauser effect
(NOE), an interaction between nuclei that is proportional to
the inverse sixth power of the distance between them.

• Magnetization is transferred from an excited nucleus to an

unexcited one if they are less than about 5 Å apart.

• In other words, the effect provides a means of detecting the

location of atoms relative to one another in the three-
dimensional structure of the protein. The diagonal of a
NOESY spectrum corresponds to a one-dimensional
spectrum. The off-diagonal peaks provide crucial new
information: they identify pairs of protons that are less than
5 Å apart
(A)An example of the data from an NMR machine. This two-dimensional NMR
spectrum is derived from the C-terminal domain of the enzyme cellulase. The spots
represent interactions between hydrogen atoms that are near neighbours in the
protein and hence reflects the distance that separates them. Complex computing
methods, in conjunction with the known amino acid sequence, enable possible
compatible structures to be derived.
(B) In (B) 10 structures, which all satisfy the distance constraints equally well, are
shown superimposed on one another, giving a good indication of the probable
three-dimensional structure
 Why So Many Structures?
• A family of related structures is generated for three reasons.

• First, not enough constraints may be experimentally accessible to

fully specify the structure.

• Second, the distances obtained from analysis of the NOESY

spectrum are only approximate.

• Finally, the experimental observations are made not on single

molecules but on a large number of molecules in solution that may
have slightly different structures at any given moment. Thus, the
family of structures generated from NMR structure analysis
indicates the range of conformations for the protein in solution.
 Problems?
• At present, NMR spectroscopy can determine the
structures of only relatively small proteins (<40
kd), but its resolving power is certain to increase.

• The power of NMR has been greatly enhanced by

the ability to produce proteins labeled uniformly
or at specific sites with 13C, 15N, and 2H with
the use of recombinant DNA technology
Circular Dichroism Spectroscopy

Protein dynamics and

secondary structure
determination
 Circular Dichroism Spectroscopy

Circular dichroism (CD) spectroscopy measures differences in

the absorption of left-handed polarized light versus right-
handed polarized light that arise due to structural asymmetry.
(measured as a quantity called mean residue whose units are
degrees cm2dmol-1)
 CD Spectroscopy
• The absence of regular structure results in zero CD intensity,
while an ordered structure results in a spectrum which can
contain both positive and negative signals.

• Chiral or asymmetric molecules produce a CD spectrum

because they absorb left and right handed polarized light to
different extents and are thus considered to be optically active.

• Biological macromolecules such as proteins and DNA are

composed of optically active elements and because they can
adopt different types of 3D structures, each type of molecule
produces a distinct CD spectrum.
 CD Spectroscopy

Different types of protein secondary structures

(helices, sheets turns and coils) give rise to different CD spectra.

Because the spectrum of a protein is directly related to its secondary structure content,
the spectrum will be the linear combination of each of these “reference” spectra,
weighted by the fraction of the type of secondary structure. Therefore it is possible to
mathematically extract the secondary structure information for an unknown protein
from its CD spectra
CD Spectroscopy Has Been Used to Monitor

• Secondary structure

• Conformational changes

• Environmental effects

• Protein folding and denaturation

• Dynamics
CD Spectrum of Recombinant IMMUTANS
• Some of the advantages of CD spectroscopy are
that it requires only very small amounts of
protein (100mg or less)

• and measurement can be done very quickly

(30 mins or less).

• But this technique will not allow the 3D

structure to be determined.
• Single Particle Cryo- EM

• Visualizing biological
complexes such as viruses,
small organelles, and
macromolecular biological
complexes of 200 kDa or
larger preserved in non-
crystaline ice
Cryo-EM analysis 31 Å resolution
GroEL-GroES Complex (Cpn60)
Structure of the S. cerivisiae
ATPase at 24Å in the detergent
Brij-35
 A Putative Respirasome

Complex III (red) Complex IV (green)

 Protein Engineering
1. Dissection of the structure and activity of existing proteins by
making systematic alterations of their structures and examining
the changes in their properties.

2. Production of novel proteins for use in medicine and industry.

3. We do not know how to design functional protein de novo, but

we can make minor alterations of existing proteins to give useful
changes in activity. We can also cut and paste segments of one
protein to another. A good example is the humanization of
monoclonal antibodies by grafting the mouse antigen-binding
loops onto human frame work allowing use in cancer treatments.
• The prerequisite for protein engineering
studies is that the enzyme has been cloned an
expressed.

• Further, unless only relatively crude

information is required, it is essential that the
structure has been solved at high resolution
 Choice of Mutation
• Choose a mutation that deletes part of a side chain or leads to an
isosteric change. Deletions are preferred to mutations that increase
the size of the side chain, especially in the interior of a protein or at
an enzyme-substrate interface. Any increase in volume of the side
chain is liable to distort the structure of the enzyme whereas small
changes in the enzyme cavity can be tolerated.

• Avoid creating buried unpaired charges. Removal of a group that

solvates a buried charge is dangerous. Solvation energies of ions
are so high that charged groups must be solvated. Unless there is
open access of solvent to buried charge residues, the structure of
mutant proteins will rearrange so that there is solvation by water
or other groups on the enzyme.
 Choice of Mutation
• Delete the minimal number of interactions. It is
difficult enough to analyze the change in just
one interaction. Avoid the deletion of multiple
interactions where possible.

• Do not add new functional groups to side chains.

The addition of a new group on a side chain can
cause local reorganization of structure if a new
group can make novel interactions
 All previous rules may be
disobeyed when appropriate!
 Choice of Mutation
• The ideal mutation is a nondisruptive deletion,
which is one which simply removes an interaction
without causing a disruption or reorganization of
a structure.

• When in doubt mutate to alanine. This is

generally a deletion mutation and alanine has few
quirks. Glycine, because of it wider freedom of
conformation can have odd effects on structure
 Random Mutagenesis
• The process of random mutagenesis is also
useful in the study of protein structure
function.

• Random mutations may be induced by PCR or

by chemical mutagenesis. This process can be
useful in identifying important residues when
detailed structural information is not available.
 Mutagenesis may also be performed at the
gross level
• Instead of changing a single amino acid residue, whole
sections of the protein may be removed by excising a
stretch of the gene. This is termed deletion mutagenesis.

• Domains of proteins may be spliced together to give

novel proteins.

• Groups can also be added to proteins to act as tags or as

aids in purification process e.g. His tags or thioredoxin.