Lecture 4: Protein Structure
and Analysis
BME2010 Research Methods
May 20, 2020
Structure – Folding –Function
and Their Relationship to
Disease
Tertiary Structure
Protein Folding
August 29, 2019
Adapted from Principles of molecular
biology at UC lecture
Proteins are important!
• The body’s building blocks.
• Second most abundant part of our bodies, compromising about
20% of our weight (70% is water).
• Muscles, organs, enzymes, antibodies…
• Often, mutations in our DNA manifest in the final protein structure.
It’s good to understand NORMAL so that we can identify and treat
when something is abnormal.
Protein Structure and Function
Tertiary Structure: How elements of
secondary structure are packed together
What’s governs the shape of the protein?
Domains
- Proteins composed of evolutionary units called domains
- Polypeptide chains containing more than 200 residues usually fold into domains
- Domains are the fundamental structural and functional unit in proteins: they can have
independent function or contribute to the function of a multidomain protein
- Domains have specific functions: such as ligand binding
- Most domains are structurally independent units that have characteristics of small globular
proteins
New Domain Combinations
- Formation of new domain combinations is an important mechanism in protein
evolution.
- Duplication is one of the main sources for creation of new proteins.
- After duplication the domain can evolve a new or modified function .
- In the domain-centric functional classification scheme domains are classified into
several categories
- Catalytic activity
- Cofactor binding
- Responsible for subcellular localization
- Protein-protein interactions
Overview of Protein Structure Hierarchy
The 3D fold (shape) of the protein
determines its function.
Secondary Structure Motifs
Secondary structure motifs are evolutionarily conserved collections of
secondary structure elements which have a defined conformation. They also
have a consensus sequence because the aa sequence ultimately determines
structure. A given motif can occur in a number of proteins where it carries
out the same or similar functions.
Some well known examples:
protein-protein
association
Ca2+/DNA binding
DNA/RNA binding
Muscle contraction
Quaternary Structure
• The arrangement of separate polypeptide chains (subunits)
into the functional protein
• Assemblies of tertiary structural units composed of homoor hetero- multimers (or oligomers)
Why have multi-domain proteins and protein complexes?
Examples of Proteins Having Quaternary Structure
HIV Protease
Potassium Channel
Protein-Protein Interactions
The
• widespread (i.e. present at all cellular levels)
• intramolecular contacts lead to protein folding
• intermolecular contacts lead to fibrils or aggregation
• aberrant protein-protein interactions have been
implicated in a number of diseases (e.g. atherosclerosis,
diabetes, Alzheimer’s, Parkinson’s, mad cow, and
Creutzfeld-Jacob)
Protein Folding
(single domain polypeptide)
Protein Folding
(single domain polypeptide)
• All information required for folding is
contained in the amino acid sequence
• Too fast to be random (folding must be
cooperative)
• Chaperone proteins help certain proteins
fold, form appropriate disulfide bonds,
interconvert prolyl cis/trans peptide bonds
Entropy favors unfolded state
Native State Stabilization
• Net stabilization of the native state conformation of a protein results
from the balance of large forces that favor both folding and unfolding
• Folding
• hydrophobic collapse (hydrophobic sidechains coalesce in the interior of the
protein structure), intramolecular hydrogen bonds, van der Waal’s
interactions
• Unfolding
• conformational entropy, hydrogen bonding to solvent (water)
Hydrophobic Effect
• Lipid molecules disperse in the solution; nonpolar
tail of each lipid molecule is surrounded by
ordered water molecules
• Lipid aggregates – Water released, surface area
reduced
• Hydrophobic groups sequestered from water -–
entropy decreased
• Ordered shell of water minimized –entropy
increased
➔
Protein Folding
• Process in which a polypeptide chain goes from a linear
chain of amino acids with vast number of more or less
random conformations in solution to the native, folded
tertiary structure
• REVIEW OF THERMODYNAMICS:
• ΔG=ΔH –TΔS
ΔG = change in Gibbs free energy
• –Negative ΔG means decrease in free energy for a process
(favorable)
• –Reaction would go spontaneously in that direction.
• • ΔH = change in enthalpy
• –reflects number and kinds of chemical bonds (including noncovalent
interactions like salt links, hydrogen bonds, and van der Waals
interactions) in reactants and products
• –MAKING bonds/interactions gives negative ΔH (favorable)
• • ΔS = change in entropy
• –increase in disorder gives positive ΔS (favorable)
ΔGfolding
• ΔGfolding (change in free energy) between unfolded structure and
folded structure is SMALL.
• ΔGfolding results from many contributions:
– enthalpy changes (ΔH): favors both folding and unfolding
• Electrostatic effects(hydrogen bonds & salt bridges)
• solvation/desolvation of charged residues
• van der Waals interactions
• steric factors
• – entropy change (ΔS)
• hydrophobic effect: favors folding
• peptide conformational entropy (degrees of freedom &
flexibility):
favors unfolding
•
• ΔGfolding results from a near balance of opposing large forces.
• Small differences in energy are important -- loss of 1 or 2
hydrogen bonds might shift equilibrium from folded state to
unfolded form of protein.
Molecular chaperones
- the great majority of proteins can fold without assistance, in a cotranslational manner
- some proteins, which may have ‘difficulties’ reaching their native
states, must be stabilized by molecular chaperones by assisted folding
- bind to nascent (emerging) polypeptides and stabilize them mostly by
binding hydrophobic residues
- otherwise these hydrophobic residues tend to associate with
other
hydrophobic residues, leading to intra- or inter-molecular
associations with other proteins that prevent proper folding
- there are dozens of different types of molecular chaperones, and some
accomplish functions different from helping protein folding
- e.g., some help protein assembly, some help to transport
proteins to various parts of the cell, some help damaged
proteins from refolding
- they do not interact with native proteins, nor do they form part of the
final folded structures.
Prion Diseases
• Prion (PRoteinaceous Infectious virON) type of infectious agent that does not
carry any genetic material
• Neurological disorders
• Mad cow disease
• Creutzfeldt-Jacob disease
• Spongiform encephalopathies
• Transmission linked to single protein (abnormal isoform of synaptic
glycoprotein)
• Majority of prion diseases occur spontaneously (linked to a single point
mutation)
• Form amyloids which disrupt the normal tissue structure
• Proteins with a pathological conformation that infect and propagate the
pathological conformation change
• Key event in pathogenesis of prion diseases is a conformational change in the
prion protein
• PrP(C) (α-helical) → PrP(Sc) (β-sheet)
β- sheet form is insoluble due to formation
of amyloid cross β structure
Amyloid fibril formation
Mechanism of formation → 3 steps:
1.
Alignment of the molecules to form β-sheets
→ fastest stage → involves H-bonds
2.
Formation of the cross-β structure → slower
than step 1 → involves Van-der-Waals forces
→ interdigitation of residues side chains ➔
“steric zipper” structure
3.
Fibril formation → involves non-covalent
bonds
Fixing Protein Misfolding
Useful Pharmacological Approaches
• Artificial chaperones
• Help the protein form the correct 3D shape
• Stabilize a partially folded form of protein to increase activity
• Facilitate post-translational modifications
• Help traffic to appropriate location
• Prevent oligomerization (Oligomer antagonists)
• Increase synthesis or decrease degradation of protein
I. Overview of proteins.
From both a chemical and structural standpoint, proteins are the most
sophisticated molecules known. They are composed of linear polymers of
amino acid joined by peptide bonds.
Classes of amino acids (see previous lectures)
Protein function results from conformation which is determined by the
sequence of the amino acids.
1. Flexibility of the peptide chain.
2. Forces that determine the conformation;
• Covalent bonds: Cys – Cys disulfide linkages.
• Electrostatic bonds (salt bridges, ionic interactions): glu— lys+.
• Hydrogen bonding: can occur between peptide bonds, terminal
amino acids and side chains. >C=0 H-N-C.
• Van de Waals forces (hydrophobic interactions).
Reminder: Spectrophotometry and Absorption!
• Spectrophotometry – method to measure how much a chemical
substance absorbs/transmits light by measuring the intensity of
light as a beam of light passes through the sample solution.
• Each compound absorbs or transmits light over a specific wavelength
range.
http://chemwiki.ucdavis.edu/Core/Physical_Chemis
try/Kinetics/Reaction_Rates/Experimental_Determi
nation_of_Kinetcs/Spectrophotometry
UV Abosrbance
A.
UV absorbance at  = 280nm (“A280”) measures the absorbance of UV light by:
•
Aromatic amino acids: Trp (=5700 M-1 cm-1) and Tyr (=1400).
•
Prosthetic groups: hemes, flavins, metal centers, etc.
•
Disulfides (=300).
The protein concentration is determined according to Beers law: A=bc
where A =absorbance
 =extinction coefficient (M-1 cm-1)
b =cuvette pathlength (1cm)
c =protein concentration
The useful form is
c=A/b
The extinction coefficient can be determined empirically using amino acid analysis
or a colorimetric assay. Alternatively, the  can be estimated by the following formula:
 = (5700 # trp)(1300 # tyr)/MW
Quantitation of proteins: UV Absorbance
• Advantages
–
–
–
–
–
Non-destructive
Fast
Direct
Reasonably sensitive (0.2-2 mg/ml)
Many buffer effects can be subtracted
• Disadvantages
– Need aromatic in the protein
– Interference by nucleic acid
– Need accurate 
Total protein content: Lowry Assay
• Lowry assay – based on the Biuret reaction and the FolinCiocalteau reaction
• Molecules with two or more peptide bonds react with Cu2+ ions in an
alkaline solution and form a purple complex (Biuret reaction). Nitrogen
atoms of the peptide bonds form a coordination bond with the metal ion.
• Reduction of the Folin-Ciocalteau reagent by tyrosine.
• Produces a strong blue color which is measured at 750nm in a
spectrophotometer.
Detection range: 0.01 to 1.0 mg/ml.
http://www.labome.com/method/Protein-Quantitation.html
Total protein content: Lowry Assay
• Advantages:
• Very Sensitive
• Well known and highly accepted
• Disadvantages:
•
•
•
•
•
Many compounds interfere
Destroys sample
Complicated, several steps
Signal becomes unstable after 30 minutes
Aromatic, high inter-protein variation
Determination of total protein content:
Bradford Assay
• Bradford assay – the Coomassie Brilliant
Blue dye binds to proteins in acidic solution
(via electrostatic and van der Waals
bonds), resulting in a shift of the absorption
maximum of the dye from 465nm to
595nm.
• Under acidic conditions, the ‘red’ form of
the dye is converted to a ‘blue’ form, as the
dye binds to the protein in the solution.
• The red form of the Coomassie dye first donates
its free electron to the ionizable group on the
protein, exposing its hydrophobic pockets.
• The ‘pockets’ bind non-covalently to the non-polar
region of the dye via van der Waals forces.
• The binding of the protein stabilizes the blue form
of the Coomassie dye.
• Detection range: 0.025 to 1.4 mg/ml.
http://www.labome.com/method/Protein-Quantitation.html
Total protein content: Bradford Assay
• Advantages:
•
•
•
•
•
Low interference by other compounds
Very Sensitive
Easy
Fast, as fast as 2 min
Good reagent stability
• Disadvantages:
• Signal is unstable after 1 hour
• Destroys sample
• High inter-protein variation
Determination of total protein content
http://www.labome.com/method/Protein-Quantitation.html
Steps/Considerations for protein
purification and analysis
(1) Choose protein to purify
(2) Choose source (natural or expressed)
Source of protein for study
Early biochemistry (1970’s)
utilized proteins that were abundant from natural sources
(myoglobin, lysozyme, hexokinase)
Middle biochemistry (1980’s to mid 1990’s)
isolated small amounts of proteins, get gene, express and
purify from bacteria, yeast, insect cells, mammalian cells
Now (2000s)
get gene from library based on homology
choose gene and express and study it
**Still problems with:
membrane proteins and solubility
Steps/Considerations for protein
purification and analysis
(2) Choose source (natural or expressed)
Break open cells by destroying membranes and releasing cytosolic protein mix crude extract
If nuclear or membrane protein - more work!
(3) Soluble in aqueous solution?? (problem with membrane proteins)
(4) Stability (perform purification/analyses in cold)
(5) Purify
Separate proteins using fractionation based on physical characteristic:
1. solubility
2. electrical charge
We’ve talked about many of these already! See the DNA lecture.
3. size + shape
4. affinity for other molecules
5. polarity
Steps/Considerations for protein
purification and analysis
(5) Purify
Characteristic:
Procedure:
Charge
1. Ion exchange chromatography
2. Electrophoresis
3. Isoelectric focusing
Size:
1. Dialysis and ultracentrifugation
2. Gel electrophoresis
3. Gel filtration (size exclusion)
chromatography
Specificity:
1. Affinity chromatography
Polarity:
1. Adsorption chromatography
2. Paper chromatography
3. Reverse-phase chromatography
4. Hydrophobic chromatography
Protein Purification and Analysis
SDS Gel Electrophoresis
Used to estimate purity and molecular weight, separate proteins
by size
Denature protein by adding SDS (then separate by size only)
SDS forms micelles
and binds to proteins
Determination of unknown protein molecular weight
Protein Purification and Analysis
Isoelectric focusing gel electrophoresis
determine the isoelectric point (pI) of a protein
separates proteins until they reach the pH that matches their pI
(net charge is zero)
Centrifugation Techniques
• Differential Centrifugation
• Gradient Centrifugation
Differential Centrifugation (Moving Zone)
• Centrifugation of a biological sample allows collection of desired materials or to
separate particles based upon their sedimentation properties
• More massive particles will sediment faster than less massive particles
• Application: quick isolation of organelles and other subcellular components
• Differential centrifugation is carried out by centrifuging a sample at low speed
and then separating the supernatant from the pellet
• The supernatant can then be re-centrifuged at a higher speed, and the
supernatant and pellet are separated again
Differential Centrifugation:
Separating Cellular Components
Low
speed
Medium
speed
High
speed
Very high
speed
Centrifugation Through Density Gradients
Rate Zonal Centrifugation
• Sample is applied in a thin layer at the top of the density gradient
• Under centrifugal force, the particles will separate/sediment through
the gradient in different zones according to their size shape and
density.
• Separation is based primarily upon size (i.e. larger particles sediment faster)
• Can separate particles of same size by their shape (ex. linear versus
globular)
• The particle with the greater frictional coefficient (f) (lesss dense)
will move slower
Centrifugation Through Density Gradients
Rate Zonal Centrifugation (cont)
• Density of the particles being separated is greater than solvent density
• Optimal centrifugation time for separating desired particles must be predetermined.
• If the centrifuge is not turned off soon enough, all of particles will pellet.
• ***This is a shared similarity with Differential Centrifugation***
Centrifugation Through Density Gradients
Isopycnic centrifugation
• Separates particles by density into zone (time-independent)
• The density gradient encompasses the whole range of densities of particles in the
sample.
• Sample can be uniformly mixed with gradient solution.
• Each particles sediments and remain at the point where gradient density is equal
to its own density.
Centrifugation Through Density Gradients
Isopycnic centrifugation (continued)
• With isopycnic gradients, the sample can also be underlaid at the bottom of the
tube and the various particles will 'float' to their correct densities during
centrifugation
Drawbacks:
• The self-generating gradient often requires long hours of centrifugation.
• Ex.) 36-48h for isopycnic banding of DNA in a CsCl gradient
• Run time (generally) cannot be shortened by increasing the rotor speed: position
of the zones in the tube since the gradient material will redistribute farther down
the tube under greater centrifugal force.
Immuno Detection of Proteins
Immuno detection (Western Blots, ELISA, solid phase, dot blots):
These methods use antibodies and are based on the following general strategy:
Step 1: Mixture of protein is adhered to a solid support (nitrocellulose, microtiter plates).
Step 2: Remaining binding sites on the solid support are blocked using BSA or dried milk.
Step 3: Primary antibody is bound to the protein of interest.
Step 4: A secondary antibody that is specific for the primary antibody is allowed to bind.
Step 5: Amount of secondary antibody bound is quantitated through the use of some marker
incorporated into the secondary antibody. (radiolabel, biotinylation, fluorescence, enzymatic products,
etc.)
Western Blot
• Western blots allow investigators to determine the molecular
weight of a protein and to measure relative amounts of the
protein present in different samples.
• Proteins are separated by gel electrophoresis, usually SDSPAGE.
• The proteins are transferred to a sheet of special blotting paper
called nitrocellulose.
• The proteins retain the same pattern of separation they had on
the gel.
Western Blot
• The blot is incubated with a generic protein (such as milk
proteins) to bind to any remaining sticky places on the
nitrocellulose.
• An antibody is then added to the solution which is able to
bind to its specific protein.
• The antibody has an enzyme (e.g. alkaline phosphatase or
horseradish peroxidase) or dye attached to it which cannot
be seen at this time.
• The location of the antibody is revealed by incubating it
with a colorless substrate that the attached enzyme
converts to a colored product that can be seen and
photographed.
Practical Notes:
• Requires specific antibodies to the protein in question.
• Cross reaction of antibodies with other or related proteins must
be carefully monitored.
• The blocking step is critical to the assay and must be
performed for at least 2 h (preferably overnight) to prevent high
backgrounds.
SDS-PAGE (PolyAcrylamide Gel
Electrophoresis)
• SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis, is a technique widely used in biochemistry,
forensics, genetics and molecular biology:
• to separate proteins according to their electrophoretic mobility
(a function of length of polypeptide chain or molecular weight).
• to separate proteins according to their size, and no other
physical feature.
SDS (sodium dodecyl sulfate) is a
detergent (soap) that can dissolve
hydrophobic molecules but also has a
negative charge (sulfATE) attached to it.
Fig.1Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing negative and positive charges due to the
charged R-groups in the protein.
The large H's represent hydrophobic domains where nonpolar R-groups have collected in an attempt to get away from the polar
water that surrounds the protein.
After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative charges which overwhelms any positive
charges the protein had due to positively charged R-groups.
The resulting protein has been denatured by SDS (reduced to its primary structure-aminoacid sequence) and as a result has been
linearized.
SDS
• SDS (the detergent soap) breaks up hydrophobic areas and
coats proteins with negative charges thus overwhelming
positive charges in the protein.
• The detergent binds to hydrophobic regions in a constant ratio
of about 1.4 g of SDS per gram of protein.
• Therefore, if a cell is incubated with SDS, the membranes will
be dissolved, all the proteins will be solubilized by the detergent
and all the proteins will be covered with many negative charges.
PAGE
•
If the proteins are denatured and put into an electric field (only),
they will all move towards the positive pole at the same rate,
with no separation by size.
• However, if the proteins are put into an environment that will
allow different sized proteins to move at different rates.
• The environment is polyacrylamide.
• the entire process is called polyacrylamide gel
electrophoresis (PAGE).
• Small molecules move through the polyacrylamide forest faster
than big molecules.
• Big molecules stays near the well.
SDS-PAGE
• The end result of SDS- PAGE has two important
features:
1) all proteins contain only primary structure
&
2) all proteins have a large negative charge which
means they will all migrate towards the positive
pole when placed in an electric field.
The actual bands are equal in size, but the proteins within
each band are of different sizes.
Sample of SDS- PAGE
Protein Sequencing
•
•
•
•
Function of protein depends on its amino acid sequence
Proteins with different functions always have different sequences
Changing just 1 amino acid can make a protein defective
Functionally similar proteins from different species have similar
sequences
Steps for sequencing a large protein:
1. Cleave S-S bonds
2. Separate subunits
3. Determine N-terminus of protein
4. Determine amino acid composition
5. Use cleavage agents to digest protein into smaller fragments
6. Amino acid composition and sequence of fragments
7. Use overlapping fragments to get full sequence
Protein Sequencing
1. Cleave disulfide bonds
To sequence large protein, first break disulfide bonds using dithothreitol (DTT).
-disulfide bonds are reduced to thiol by DTT
Protein Sequencing
2. Separate subunits
Denaturation = loss of 3D structure resulting in loss of function
Denaturation affects weak interactions, such as H-bonds
Denature proteins by:
Heat, extreme pH, add organics (alcohol, acetone)
Add urea, guanidine hydrochloride, detergent
Separate subunits by gel electrophoresis, chromatography, etc.
Protein Sequencing
3. Determine N-terminus of protein
Protein Sequencing
4. Determine amino acid composition
6 M HCl
heat
Free amino
acids
HPLC or Ion-exch.
chromatography
AA
composition
Polypeptide
Determine types and
amounts of amino
acids
FDNB
+ Free
amino acids
Identify amino-terminal
residue of protein
6 M HCl
2,4-Dinitrophenyl
derivative
of amino-terminal AA
2,4-Dinitrophenyl
derivative
of protein
Determine number
of polypeptides
Phenylisothiocyanate
+
Trifluoro
acetic
acid
Phenylisothio
Cyanate
derivative of
aminoterminal AA
Identify amino-terminal
residue of protein
Purify and recycle
remaining peptide
fragment through Edman
process
Protein Sequencing
5. Use cleavage agents to digest protein into smaller fragments
Proteases
Chemical
Protein Sequencing
6. Amino acid composition and sequence of fragments
7. Use overlapping fragments to get full sequence
Protein Sequencing – What does it tell us?
Clues about functions of proteins/role of specific sequences
Elucidate history of life on earth
Macromolecular Structure
ATOMS
MOLECULES
C-C bond
ASSEMBLIES
CELLS
Resolution
limit of light
microscope
Red blood
cell
Hemoglobin
Glucose
1Å
10-10 m
MACROMOLECULES
10 Å
10-9 m
1 nm
X-ray
crystallography,
Solution NMR
Ribosome
102 Å
10-8 m
103 Å
10-7 m
Electron
microscopy
Bacterium
104 Å
10-6 m
1 µm
105 Å
10-5 m
Fluorescence spectroscopy
Step 1 : Excitation: A photon of energy is supplied by an external source (ie
UV lamp or laser) and absorbed by the fluorophore, creating an excited
electronic singlet state (S1')
Step 2: Excited State Lifetime is Finite: Typically 1-10 ns. During this time,
the fluorophore undergoes conformational changes and is subject to many
possible interactions with its molecular environment.
Step 3 : Fluorescence Emission: A photon of energy is emitted, returning
the fluorophore to its ground state S0. Due to energy dissipation during the
excited-state lifetime, the energy of this photon is lower, and therefore has a
longer wavelength than the excitation photon.
The difference in energy or wavelength represented by is called the Stokes
shift. The Stokes shift is fundamental to the sensitivity of fluorescence
techniques because it allows emission photons to be detected against a low
background, isolated from excitation photons. In contrast, absorption
spectrophotometry requires measurement of transmitted light relative to high
incident light levels at the same wavelength.
Fluorescence spectroscopy
hv
Fluorescence spectroscopy - Benefits
Sensitivity: Detectability to parts per billion or even parts per trillion is
common for most analytes. This extraordinary sensitivity allows reliable
detection of fluorescent materials using small sample sizes.
Fluorometers achieve 1,000-500,000x better limits of detection as
compared to other commonly used spectrometers.
Specificity: Spectrophotometers merely measure absorbed light, and
are prone to interference problems because many materials absorb
light, making isolating the targeted analyte in a complex matrix difficult.
Fluorometers are highly specific and less susceptible to interferences
because fewer materials absorb and also emit light (fluoresce). And if
non-target compounds do absorb and emit light, it is rare that they will
emit the same wavelength of light as target compounds.
Wide Concentration Range: Fluorescence output is linear to sample
concentration over a very broad range.
Fluorescence spectroscopy Drawbacks
Alteration of analyte: Attachment of fluorescently labeled probes in
the protein may create conformational changes in the molecule.
Buffer effects: Scattering, collisional relaxation, quenching
wavelength overlap.
Contaminants with any auto fluorescence cause major problems.
Fluorescence spectroscopy
X-Ray Crystallography
Crystal -> Diffraction pattern -> Electron density -> Model
Macromolecular Structure
•
•
•
•
•
•
X-ray crystallography
Determines 3-dimensional structure of a protein.
Need a lot of pure protein in crystallized form
Based on Bragg’s Law (use X-ray diffraction to understand 3-D
structure)
Beam of X-rays of given wavelength which is diffracted by electrons
of atoms in protein
Collect diffracted x-rays on photographic film
Create electron density map using Fourier transform
X-Ray Crystallography
The “gold standard”of protein structure determination.
Step 1: Purified proteins are induced to form crystals of larger than 0.5
mm in size. This is critically dependent on several parameters
including: pH, temperature, protein concentration, solvent and ionic
strength. {This is the most difficult step}
Step 2: The crystal is bombarded with X-rays which are diffracted by
the electron dense proteins in the crystal. These diffracted X-rays are
then detected by an area detector. The intensity, amplitude and phase
of the rays are fed into a computer.
Step 3 Analysis: The data is used to compile an electron density map
which, using the primary sequence of the protein, is converted into a
structural model by computer.
X-Ray Crystallography
Benefits
• Most accurate detailed structural information and highest resolution.
• Can obtain secondary structural information and tertiary folding information
of proteins.
X-Ray Crystallography
Drawbacks
• Many proteins are difficult to crystallize and usually require lots of protein
• Not very useful in studying dynamics since the proteins are studied under
crystalline state
• May not be as same as the solution structure.
• Expensive machinery (collaborate)
Nuclear Magnetic Resonance
• NMR is a powerful tool available for organic
structure determination.
• It is used to study a wide variety of nuclei:
– 1H
– 13C
– 15N
– 19F
– 31P
Nuclear Spin
• A nucleus with an odd atomic number or
an odd mass number has a nuclear spin.
• The spinning charged nucleus generates
a magnetic field.
78
Two Energy States
The magnetic fields of
the spinning nuclei
will align either with
the external field, or
against the field.
A photon with the right
amount of energy
can be absorbed
and cause the
spinning proton to
flip.
=>
Protons in a Molecule
Depending on their chemical environment, protons in a molecule
are shielded by different amounts.
NMR Signals
• The number of signals shows how many
different kinds of protons are present.
• The location of the signals shows how
shielded or deshielded the proton is.
• The intensity of the signal shows the
number of protons of that type.
• Signal splitting shows the number of
protons on adjacent atoms.
The NMR Spectrometer
The NMR Graph
Nuclear Magnetic Resonance
Benefits
• Non-destructive solution structure determination method
• Most information content based on a solution technique
• Can extract 3D structural information secondary structural
information
• Useful for studying molecular dynamics
Drawbacks
• Need high concentrations up to several mgs.
• Extremely expensive machinery
• May require isotope enrichment
• Time consuming data analysis, resonance assignments can take up
to several months
Macromolecular Structure
• Circular dichroism (CD) spectroscopy
• Provides basic information on the overall secondary structure of a
protein, including the percentage of beta sheets and alpha helices.
• Measures differences in the absorption of left- and right-handed
polarized light that arises from asymmetric structures.
• Usually carried out in the far-UV spectrum.
http://web.nmsu.edu/~kburke/Instrumentation/CD1.html
http://www.fbs.leeds.ac.uk/facilities/cd/
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/proteinbiology-resource-library/pierce-protein-methods/overview-protein-protein-interaction-analysis.html
Immunoprecipitation
• An antibody against a specific target
protein forms an immune complex
with the target in a sample (cell
lysate).
• The immune complex is then
captured, or precipitated, on a beaded
support to which an antibody-binding
protein is immobilized (Protein A or
G).
• Any proteins not precipitated on the
beads are washed away.
• The antigen/antibody is eluted and
analyzed via SDS-PAGE, followed by
Western blot to verify the identity of
the antigen.
https://www.thermofisher.com/us/en/home/life-science/proteinbiology/protein-biology-learning-center/protein-biology-resourcelibrary/pierce-protein-methods/co-immunoprecipitation-co-ip.html
Co-Immunoprecipitation
• An extension of
immunoprecipitation.
• IP reaction captures/purifies the
primary target and other bound
macromolecules.
https://www.thermofisher.com/us/en/home/life-science/proteinbiology/protein-biology-learning-center/protein-biology-resourcelibrary/pierce-protein-methods/co-immunoprecipitation-co-ip.html