Download MBBS (Bachelor of Medicine, Bachelor of Surgery) 1st year (First Year) Biochemistry ppt lectures Topic 25 Protein Structure Determination Notes. - biochemistry notes pdf, biochemistry mbbs 1st year notes pdf, biochemistry mbbs notes pdf, biochemistry lecture notes, paramedical biochemistry notes, medical biochemistry pdf, biochemistry lecture notes 2022 ppt, biochemistry pdf.
Protein Structure Determination
Specific learning objectives
? Differential centrifugation used to prepare subcellular fractions or to isolate
specific organelles.
? Purification methods are based on properties of proteins.
? Methods of Proteins Purification includes chromatography techniques.
? Protein denaturation and renaturation.
Introduction
? First step in protein purification is to break and open these cells, releasing their
proteins into a solution called a crude extract.
? Differential centrifugation can be used to prepare subcellular fractions or to
isolate specific organelles.
? Once the extract or organel e preparation is ready, various methods are available
for purifying one or more of the proteins.
Differential Centrifugation
Fig1.8 a: Lehninger Principles of Biochemistry by David L Nelson
Methods of Proteins Purification
All purification techniques are based on:
? Stability
? Solubility
? Size
? Charge
? Binding affinity
? Hydrophobicity
Chromatographic techniques separate one protein from another based upon
difference in their:
? Size (size exclusion chromatography),
? Charge (ion-exchange chromatography),
? Hydrophobicity (hydrophobic interaction chromatography),
? Ability to bind a specific ligand (affinity chromatography).
Dialysis
? Proteins separated from small molecules such as salt by dialysis through a
semipermeable membrane with pores.
? Protein mixture is placed inside the dialysis bag, submerged in a buffer solution
that devoid of small molecules to be separated away.
? Molecules dimensions significantly greater than the pore diameter are retained
inside the dialysis bag.
Cont--
? Smaller molecules and ions
capable of passing through the
pores of the membrane diffuse
down
their
concentration
gradients and emerge in the
solution outside the bag.
? Useful for removing a salt or other
small molecule from a cell
fractionate, but it will not
distinguish between proteins
effectively.
Fig. 3.2. Protein molecules (red) are retained within the dialysis bag, whereas smal molecules
(blue) diffuse into the surrounding medium. Biochemistry 7th edition by Jeremy M. Berg, John
L. Tymoczko and Lubert Stryer.
Hydrophobic Interaction Chromatography
? It separates proteins based on their tendency to associate with a stationary phase
matrix coated with hydrophobic groups (eg, phenyl Sepharose, octyl Sephadex).
? Proteins with exposed hydrophobic surfaces adhere to the matrix via hydrophobic
interactions enhanced by employing a mobile phase of high ionic strength.
Cont--
? After non-adherent proteins are washed away, polarity of the mobile phase is
decreased by gradually lowering its salt concentration.
? If the interaction between protein and stationary phase is strong, ethanol or
glycerol added to the mobile phase to decrease its polarity and weaken
hydrophobic interactions.
Short hydrophobic-interaction chromatography video attached with email
Column Chromatography
? Porous solid material with appropriate chemical properties (the stationary phase)
held in a column, and a buffered solution (the mobile phase) pass through it.
? Protein-containing solution, layered on the top of the column, pass through the
solid matrix as an ever-expanding band within the larger mobile phase.
? As the mobile-phase liquid emerges from the column, it is automatically collected
in a series of small portions called fractions.
Column Chromatography
Column chromatography: The standard elements of a chromatographic column include a solid, porous material supported inside a column, general y made of plastic or glass. The solid material (matrix) makes up the
stationary phase through which flows a solution, the mobile phase. The solution that passes out of the column at the bottom (the effluent) is constantly replaced by solution supplied from a reservoir at the top. The
protein solution to be separated is layered on top of the column and al owed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a band within the mobile phase that is
initial y the depth of the protein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. The overal protein
band thus widens as it moves through the column. Individual types of proteins (such as A, B, and C, shown in blue, red, and green) gradual y separate from each other, forming bands within the broader protein band.
Separation improves (resolution increases) as the length of the column increases. However, each individual protein band also broadens with time due to diffusional spreading, a process that decreases resolution. In this
example, protein A is wel separated from B and C, but diffusional spreading prevents complete separation of B and C under these conditions. Fig 3.17: Lehninger Principles of Biochemistry by David L Nelson
Short column chromatography video attached with email
High-Pressure Liquid Chromatography (HPLC)
? Column materials are very finely divided and, as a consequence, more interaction
sites and greater resolving power.
? Column is made of finer material, high-pressure applied to the column to obtain
adequate flow rates.
? Net result is both high resolution and rapid separation.
Cont--
? In a typical HPLC setup, a detector that monitors the absorbance of the eluate at
a particular wavelength is placed after the column.
? In the sample HPLC elution profile, proteins are detected by setting the detector
to 220 nm (the characteristic absorbance wavelength of the peptide bond).
? In a short span of 10 minutes, a number of sharp peaks representing individual
proteins can be readily identified.
Cont--
Gel filtration by HPLC clearly defines
the individual proteins because of its
greater resolving power:
1. Thyroglobulin (669 kd),
2. Catalase (232 kd),
3. Bovine serum albumin (67 kd),
4. Ovalbumin (43 kd), and
5. Ribonuclease (13.4 kd).
Fig. 4.6. High-Pressure Liquid Chromatography (HPLC): Biochemistry 7th edition by Jeremy M.
Berg, John L. Tymoczko and Lubert Stryer.
Short HPLC video attached with email
Ion-Exchange Chromatography
? In mobile phase, proteins with a net positive charge migrate through the matrix
more slowly than those with a net negative charge, because the migration of the
former is retarded more by interaction with the stationary phase.
? Expansion of the protein band in mobile phase caused both by separation of
proteins with different properties and by diffusional spreading.
Cont--
? As the length of the column increases, the resolution of two types of protein with
different net charges generally improves.
? Rate at which the protein solution can flow through the column decreases with
column.
Ion-Exchange Chromatography
Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH. The column matrix is a synthetic polymer containing bound charged groups; those with
bound anionic groups are cal ed cation exchangers, and those with bound cationic groups are cal ed anion exchangers. Ion-exchange chromatography on a cation exchanger is shown here. The affinity of each protein for the
charged groups on the column is affected by the pH (which determines the ionization state of the molecule) and the concentration of competing free salt ions in the surrounding solution. Separation can be optimized by
gradual y changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient. Fig 3.18 a: Lehninger Principles of Biochemistry by David L Nelson. Short Ion-exchange video attached with email
Affinity Chromatography
? Based on binding affinity of a protein.
? Beads in column covalently attached to chemical group.
? Protein with affinity for this particular chemical group bind to the beads in the
column, and its migration retarded.
Affinity Chromatography
Affinity chromatography separates proteins by their binding specificities. The proteins retained on the column are those that bind specifical y to a ligand cross-linked to the beads. (In biochemistry, the term "ligand" is used to
refer to a group or molecule that binds to a macromolecule such as a protein.) After proteins that do not bind to the ligand are washed through the column, the bound protein of particular interest is eluted (washed out of the
column) by a solution containing free ligand. Fig 3.18 c: Lehninger Principles of Biochemistry by David L Nelson.
Short affinity-chromatography video attached with email
Gel-Filtration/Molecular Exclusion Chromatography
? More-discriminating separations on the basis of size.
? Sample is applied to the top of a column consisting of porous beads made of an
insoluble but highly hydrated polymer such as dextran or agarose (which are
carbohydrates) or polyacrylamide.
? Sephadex, Sepharose, and Biogel are commonly used for preparations of these
beads (0.1 mm).
? Small molecules can enter these beads, but large ones cannot.
Cont--
? Small molecules distributed in the aqueous solution both inside the beads and
between them, whereas large molecules located only in solution between the
beads.
? Large molecules flow more rapidly through this column and emerge first because
a smaller volume is accessible to them.
? Molecules enter a bead will flow from the column at an intermediate position,
and small molecules, which take a longer path, exit last.
Gel-Filtration Chromatography
Fig. 4.3. Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer.
Short gel-filtration chromatography video attached with email
Denaturation at the protein levels
At primary structure: Sequence of aa held together by covalent peptide bonds, is
not disrupted by denaturation.
At secondary structure: Proteins lose all regular repeating patterns such as -
helices and -pleated sheets and adopt a random coil shape.
Cont--
At tertiary structure: Disruption of covalent interactions between aa side chains
(such as disulfide-bridges bet cysteine groups), non-covalent interactions between
polar aa side-chains, van-der waals interactions between non-polar aa side chains.
At quaternary structure: Protein sub-units are dissociated and/or spatial
arrangement of protein subunits is disrupted.
Cont--
? Native Protein denaturate in presence of reducing agent, alter pH, temp, ionic strength,
and solubility.
? Denature protein causes loss of structure and function.
Various denaturants are:
1. Heat disrupts hydrogen bonds and hydrophobic interactions bet non-polar residues. Ex.
Albumin in egg denature and coagulate during cooking.
1. Strong acids and bases disrupts the salt bridges formed in a protein structure. Ex. In
digestive system, acidic gastric juices causes coagulation of milk by proteolytic enzyme
renin.
Cont--
2. Urea disrupts the stabilizing hydrophobic interactions, thus freeing entire
polypeptide from its folded conformation.
3. Reducing agents like guanidine hydrochloride (GdnHCL) or -mercaptoethanol
reduces the disulphide bonds to sulfhydryl group and breaks intra and interchain
disulphide bonds.
4. Detergents (SDS) disrupts hydrophobic interactions.
Renaturation of Unfolded Proteins
? The Nobel prize in chemistry 1972 was divided to Christian B Anfinsen provided
the first evidence that:
Aa sequence of a polypeptide chain contains all information required to fold the
chain into its native, 3-D structure.
? Certain globular proteins denatured by heat, extremes of pH, or denaturing
reagents will regain their native structure and their biological activity if returned
to conditions in which the native conformation is stable. This process is called
renaturation.
Renaturation of Unfolded, Denatured Ribonuclease
Lehninger Principles of Biochemistry by David L Nelson
Summary
? Proteins are separated and purified based on their properties.
? Proteins precipitated by the addition of certain salts.
? Chromatographic procedures makes use of differences in size, binding affinities,
charge, and other properties.
? These include ion-exchange, size-exclusion, affinity, and high-performance liquid
chromatography.
Cont--
? The 3-D structure and the function of proteins destroyed by denaturation, which
demonstrate the relationship between structure and function.
? Some denatured proteins can renature spontaneously to form biologically active
protein.
Reference Books
1) Harper 's Il ustrated Biochemistry-30th edition.
2) Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer.
3) Lehninger Principles of Biochemistry by David L Nelson.
4) Short you-tube video clips on types of chromatography.
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This post was last modified on 05 April 2022