Unit 1: Structure Determination

Fundamentals of X-ray Crystallography

NMR Spectroscopy of Marcomolecules

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Levels of Protein Structures

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 Protein Sequencing
• Edman degradation is the process of purifying protein by
sequentially removing one residue at a time from the amino end of
a peptide.
• To solve the problem of damaging the protein by hydrolyzing
conditions, Pehr Edman created a new way of labeling and cleaving
the peptide.
• Edman thought of a way of removing only one residue at a time,
which did not damage the overall sequencing.
• This was done by adding Phenyl isothiocyanate, which creates a
phenylthiocarbamoyl derivative with the N-terminal. The N-
terminal is then cleaved under less harsh acidic conditions, creating
a cyclic compound of phenylthiohydantoin (PTH) - amino acid.
• This does not damage the protein and leaves two constituents of
the peptide.
• This method can be repeated for the rest of the residues,
separating one residue at a time.
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Edman Cycle

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 Sequencing Larger Proteins
• Larger proteins cannot be sequenced by the Edman sequencing because of the
less than perfect efficiency of the method.
• A strategy called divide and conquer successfully cleaves the larger protein into
smaller, practical amino acids.
• This is done by using a certain chemical or enzyme which can cleave the protein at
specific amino acid residues.
• The separated peptides can be isolated by chromatography.
• Then they can be sequenced using the Edman method, because of their smaller
size.
• In order to put together all the sequences of the different peptides, a method of
overlapping peptides is used.
• The strategy of divide and conquer followed by Edman sequencing is used again a
second time, but using a different enzyme or chemical to cleave it into different
residues.
• This allows two different sets of amino acid sequences of the same protein, but at
different points.
• By comparing these two sequences and examining for any overlap between the
two, the sequence can be known for the original protein.

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 Peptide
Gly Pro Thr Gly Arg Leu Glu Ser Lys Cys Met Val

Digestion using chemicals or enzyme

Gly Pro Thr Gly Arg

Perform Edman degradation

Thr Gly Arg Leu Glu Ser for every fragment individually

Glu Ser Lys Cys Met Val

Assemble the sequences of

Gly Pro Thr Gly Arg individual fragment to get consensus sequence

Thr Gly Arg Leu Glu Ser

Glu Ser Lys Cys Met Val

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 Protein Secondary Structure
• Local structures of a protein with regular
conformations are known as secondary
structures.
• They are stabilized by hydrogen bonds formed
between carbonyl oxygen and amino
hydrogen of different amino acids.
• Chief elements of secondary structures are α-
helices and β-sheets.

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α-helices and β-sheets

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 α-helices
• α-helices are right handed, exhibiting a rightward spiral
form and contains 3.6 amino acids per helical turn.
• The structure is stabilized by hydrogen bonds formed
between the main chain atoms of residues i and i + 4.
• The hydrogen bonds are nearly parallel with the helical
axis.
• Hydrophobic residues of the helix tend to face inside and
hydrophilic residues of the helix face outside.
• Thus, every third residue along the helix tends to be a
hydrophobic residue.
• Ala, Gln, Leu, and Met are commonly found in an α-helix,
but not Pro, Gly, and Tyr.
• These rules are useful in guiding the prediction of protein
secondary structures.

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 β-Sheets
• The β-strand conformation is pleated with main chain backbone
zigzagging and side chains positioned alternately on opposite
sides of the sheet.
• β-Strands are stabilized by hydrogen bonds between residues of
adjacent strands
• β-strands near the surface of the protein tend to show an
alternating pattern of hydrophobic and hydrophilic regions,
whereas strands buried at the core of a protein are nearly all
phydrohobic.
• The β-strands can run in the same direction to form a parallel
sheet or can run every other chain in reverse orientation to
form an antiparallel sheet, or a mixture of both.
• The hydrogen bonding patterns are different in each
configurations.
• Because of the long-range nature of residues involved in this
type of conformation, it is more difficult to predict β-sheets
than α-helices.
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 Coils and Loops
• There are also local structures that do not belong to regular
secondary structures (α-helices and β-strands).
• The irregular structures are coils or loops.
• The loops are often characterized by sharp turns or hairpin-like
structures.
• If the connecting regions are completely irregular, they belong
to random coils.
• Residues in the loop or coil regions tend to be charged and
polar and located on the surface of the protein structure.
• They can be functionally significant because these locations are
often the active sites of proteins.
• Coiled coils are a special type of supersecondary structure
characterized by a bundle of two or more α-helices wrapping
around each other.
• The helices forming coiled coils have a unique pattern of
hydrophobicity, which repeats every seven residues (five
hydrophobic and two hydrophilic).

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 Secondary Structure Prediction isn’t
working
• Prediction quality has not improved much even with
the huge growth of training data
• Secondary structures are not completely determined
by local forces
• Long distance interactions do not appear in sliding
window
• Empirical studies show that same amino acid
sequences can assume multiple secondary structures

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 Structure Prediction
• Conventional techniques
– X-Ray Crystallography
– NMR techniques
• Experimental methods used to determine protein structures
are time consuming and limited in their approach.
• Currently, it takes 1 to 3 years to solve a protein structure.
• Certain proteins, especially membrane proteins, are
extremely difficult to solve by x-ray or NMR techniques.

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Protein Expression and Purification

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Protein Crystallization Methods

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Steps in Structure determination using
X-Ray Crystallography

• First is production and the purification of the

macromolecule, its crystallization, diffraction data
collection and processing.
• Second is determination of the phases of the measured
signal, absolutely required to calculate the electron
density.
• The last step is the refinement of the built structure,
called the model, which will then be interpreted in the
context of its biological function.

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 X-Ray Crystallography
• The most accurate technique used to obtain structure coordinates for
proteins is called X-Ray Crystallography.
• In X-ray crystallography, a crystal of a substance is placed in an X-ray
beam.
• X-rays are reflected by the electron clouds surrounding the atoms in
the crystal.
• In a protein crystal, individual protein molecules are arranged in a
regular lattice, so X-rays reflected by the crystal in regular pattern.
• The X-ray reflections scattered from a protein crystal can be analyzed
to produce an electron density map of the protein.
• Protein atomic coordinates are produced by modeling the possible way
for the atoms making up the known sequence of the protein to felt
into this electron density.
• Two points to remember
– Protein sample must be pure protein sample.
– The protein sample has to form crystals that are relatively large and singular.

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X-ray crystallographic workflow

Phasing
Failure

Built Atomic Model Electron Density

Model Evaluation,
Refinement
to Final Structure
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Final Structure from
X-ray Crystallography

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 Why use NMR?
• Can’t get a crystal / want to work in solution
• Want to look at binding to other
proteins/molecules
• Want to understand stability
• Want to measure fast dynamics processes

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 What is NMR?
• Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical
chemistry technique used for determining the content and purity
of a sample as well as its molecular structure.
• NMR can quantitatively analyze mixtures containing known
compounds.
• For unknown compounds, NMR can either be used to match against
spectral libraries or to infer the basic structure directly.
• Once the basic structure is known, NMR can be used to determine
molecular conformation in solution as well as studying physical
properties at the molecular level such as conformational exchange,
phase changes, solubility, and diffusion.
• An increasing number of protein structure are being solved by
Nuclear Magnetic resonance (NMR) spectroscopy.
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 The basis of NMR
• The principle behind NMR is that many nuclei have
spin and all nuclei are electrically charged.
• If an external magnetic field is applied, an energy
transfer is possible between the base energy to a
higher energy level (called as shifting o nuclei).
• The energy transfer takes place at a wavelength that
corresponds to radio frequencies and when the spin
returns to its base level, energy is emitted at the same
frequency.
• By interpreting the chemical shifts observed in the
NMR spectrum of a molecule, distances between
particular atoms in the molecule can be estimated.

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 NMR Block Diagram

It consist of the following components, a powerful magnet with a homogeneous

field that can be continuously and precisely varied over the relative narrow range;
this is accomplished by a sweep generator. A radiofrequency transmitter and a radio-
frequency receiver are the two other components of the spectrometer. Besides
these, the instrument has a recorder, a calibrator and an integrator.
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 Example
• In the case of the 1H NMR spectrum of ethyl benzene, the methyl (CH3)
group is the most electron withdrawing (electronegative) and therefore
resonates at the lowest chemical shift.
• The aromatic phenyl group is the most electron donating
(electropositive) so has the highest chemical shift.
• The methylene (CH2) falls somewhere in the middle. However, if the
chemical shift of the aromatics were due to electropositivity alone, then
they would resonate between four and five ppm.
• The increased chemical shift is due to the delocalized ring current of the
phenyl group.
• This definition of chemical shift is sufficient for most purposes. However,
complications arise when comparing chemical shifts under different
conditions: solvent, temperature, etc. Chemical shifts are affected
slightly by isotopic substitution, an effect that is known as an isotope
shift. 26
1H NMR spectrum of ethylbenznene
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 1D 1H NMR spectrum of a small
protein
• For even small
proteins, 1D
spectra are
complicated and
cannot be analyzed
comprehensively
• 1D spectra can be
useful, however, for
evaluating the
suitability/stability
of a protein sample

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Final Structure from NMR

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 X-ray crystallography vs NMR

• For proteins that cannot

• For proteins that can
form crystals
crystallize
• Used for smaller proteins
• Used for larger
proteins • Proteins mixtures can
also be analyzed
• Proteins should be
> 95% pure • Final structure is consists
of multiple
• Final structure is only
representations of the
one
protein
• Dynamics cannot be
• Dynamics can be
observed
observed

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