Chapter 3- Exploring proteins Flashcards
Proteome
Derived from proteins expressed by the genome. It encompasses the types, functions, and interactions of proteins within its biological environment. Only a subset of the proteins encoded by genes will actually be present in a given biological context
Is a proteome a fixed characteristic of the cell?
No, it represents the functional expression of information, and it varies with cell type, developmental stage, and environmental conditions. It is highly dynamic
Why is the proteome larger than the genome?
Almost all gene products are proteins that can be chemically modified in a variety of ways
Purification
Should yield a sample containing only one type of molecule
Assay
A test. Biochemists use assays for some unique identifying property of the protein to monitor the success of purification. A positive result on the assay indicates that the protein is present. The more specific the assay, the more effective the purification
How are enzymes detected in a sample?
The assay usually measures enzyme activity, or the ability of the enzyme to promote a specific chemical reaction. This is done indirectly. Lactate dehydrogenase activity is measured by examining how much light absorbing ability is developed by the sample in a given period of time. Molecules produced by the reaction have differing abilities to absorb light
Homogenate
A mixture of all of the components of the cell, after the cell has been disrupted in a homogenizer
What has to happen to the cell before the protein can be purified?
The protein has to be released from the cell. A homogenate is formed by disrupting the cell membrane, and the mixture is fractionated by centrifugation and different proteins can be separated based on density
Differential centrifugation
The mixture is fractionated by a centrifuge, and creates a dense pellet of heavy material at the bottom of the tube with a lighter supernatant above. The supernatant can then be centrifuged at a greater force to create another pellet and supernatant. It yields multiple fractions of decreasing density, with each one containing hundreds of proteins. The fractions are each separately assayed for the desired activity
Salting out
Most proteins are less soluble at high salt concentrations. The salt concentration at which a protein precipitates differs from one protein to another. This means that salting out can be used to fractionate proteins and to concentrate dilute solutions of proteins
Dialysis
Uses a semipermeable membrane (like cellulose with pores) to separate proteins from small molecules like salt. The protein mixture is placed inside the dialysis bag, which is then submerged in a buffer solution that lacks the small molecules to be separated away. Molecules with dimensions greater than that of the pore diameter are left inside the bag, and smaller molecules and ions can pass through the pores and leave the bag, moving down their concentration gradient. It separates small molecules from a cell fractionate, but does not distinguish between proteins
Gel-filtration (molecular exclusion) chromatography
Separates proteins on the basis of size. The sample is added to the top of a column consisting of porous beads made of an insoluble polymer. Small molecules can enter the beads, but large ones can’t. The small molecules are distributed inside the beads and between them, but large molecules flow more rapidly through the column and emerge first. Molecules of medium size will occasionally enter the beads and will leave at an intermediate position, while small molecules take a longer path and exit last
Ion-exchange chromatography
Separates proteins on the basis of their net charge. A protein with a net positive charge will usually bind to a column of beads containing carboxylate groups. A negatively charged protein will not. The bounds protein can then be eluted (released) by increasing the concentration of sodium chloride in the eluting buffer. Sodium ions compete with positively charged groups on the protein for binding to the column. Proteins that have a low density of net positive charge will tend to emerge first (those with the same charge as the column), followed by those with a higher charge density
Cation exchange
Another name for ion exchange chromatography. It indicates that positively charged groups will bind to the anionic beads. Positively charged proteins are considered cationic and are separated by chromatography on negatively charged CM-cellulose columns.
Anion exchange
Negatively charged (anionic) proteins are separated on positively charged DEAE-cellulose columns.
Affinity chromatography
Takes advantage of the high affinity of proteins for specific chemical groups. The protein will pass through a column of beads containing residues of the compound with the affinity for that protein. This is a technique that can be used to isolate transcription factors. The protein mixture is passed through a column that contains specific DNA sequences attached to a matrix. Proteins with a high affinity for the sequence will bind and be retained. The transcription factor is released by washing with a solution that has a high concentration of salt, or with a solution that has a high concentration of the compound to which the protein is bound
Transcription factors
Proteins that regulate gene expression by binding to specific DNA sequences
High performance liquid chromatography
In column chromatography, a solvent drips through a column filled with an adsorbent under gravity. HPLC is a highly improved form of column chromatography. A pump forces a solvent through a column under high pressures. The column is made of finer materials, so they have more interaction sites and more resolving power. Pressure, using high pressure pumps, has to be applied to the column to obtain adequate flow rates. This causes high resolution and rapid separation. The components of a mixture are separated from each other due to their different degrees of interaction with the absorbent particles. This causes different elution rates for the different components and leads to the separation of the components as they flow out the column.
Gel electrophoresis
Uses an electric field to separate molecules with net charges, like proteins, DNA, and RNA. It is performed in a thin, vertical slab of polyacrylamide gel. All molecules, regardless of size, are forced to move through the gel due to the electric field. The proteins are prepared using SDS. Proteins in the sample are separated on the basis of mass. Small proteins move through the gel rapidly, but large proteins stay near the top of the gel.
Why is polyacrylamide gel used in gel electrophoresis?
The gels are chemically inert and readily formed by the polymerization of acrylamide with a small amount of the cross linking agent methylenebisacrylamide to make a 3D mesh.
SDS
Before electrophoresis, the proteins are dissolved in a solution of SDS, which is an anionic detergent that disrupts almost all noncovalent interactions in the proteins. Beta-mercaptoethanol is also used to reduce disulfide bonds. Anions of SDS bind to the main protein chains and give the protein a negative charge that is much greater than the charge of the native protein, but the charge is proportional to the mass of the denatured protein
How are proteins visualized in gel electrophoresis?
They are visualized by staining with silver nitrate or Coomassie blue dye, which creates a series of bands
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Rapid, sensitive, and capable of a high degree of resolution. Even a very small amount of a protein can be stained and create a band. Proteins that differ in mass by about 2% can usually be distinguished with SDS-PAGE. SDS denatures proteins, and 1 molecule of SDS binds for every 2 amino acids, giving every protein the same charge-mass ratio so all proteins will move on the basis of mass only
Velocity of proteins migrating in an electric field
v= Ez/f
E is electric field strength, z is the net charge on the protein, and f is the frictional coefficient. V is inversely proportional to f
Frictional coefficient of proteins migrating in an electric field
f= 6(pi)(eta-n)(r)
Eta (n) is the viscosity of the medium
r is the radius of a sphere
Electrophoretic mobility
Increases for more highly charged solutes and for solutes of smaller size. The electrophoretic mobility of many proteins in SDS–polyacrylamide gels is inversely proportional to the logarithm of their mass. The graph has a negative slope and is linear
Isoelectric point
The pH at which the protein’s net charge is zero. At this pH, the electrophoretic mobility is zero
Isoelectric focusing
Separating proteins on the basis of their isoelectric point. Each protein will move until it reaches a position in the gel at which the pH is equal to the pI of the protein, or the point at which their charge is zero. In electrophoresis, a molecule will always stop moving once it has no charge. This method can resolve proteins that differ in pI by as little as .01, so proteins differing by one net charge can be separated.
How is the pH gradient formed in the isoelectric focusing gel?
By subjecting a mixture of polyampholytes (small multicharged polymers) having many different pI values to electrophoresis
Amino acids containing carboxyl groups are uncharged when
Protonated- Asp and Glu are examples
Amino acids containing nitrogen groups are uncharged when
Deprotonated- Lys, His, and Arg are examples
Two dimensional electrophoresis
Isoelectric focusing combined with SDS-PAGE. A sample is first subjected to isoelectric focusing. The single lane gel is placed horizontally on top of an SDS-polyacrylamide slab. The proteins are therefore spread across the top of the polyacrylamide gel according to how far they migrated during isoelectric focusing (on the basis of their isoelectric point). Then, they undergo electrophoresis again in a vertical direction to yield a 2D pattern of spots. The proteins spread in the vertical direction on the basis of mass
2D gel electrophoresis is typically used for
Proteins isolated from cells under different physiological conditions. The intensities of the individual spots on the gels can be compared, indicating the concentrations of specific proteins have changes in response to the physiological state
How is a protein purification scheme quantitatively evaluated? (5)
Measured by calculating the specific activity after each protein purification technique. The sample then undergoes SDS-PAGE. The techniques analyzed includes homogenization, salt fractionation, ion exchange chromatography, gel filtration chromatography, and affinity chromatography. Specific activity should increase with each step of the purification procedure since total protein is being removed while desired enzyme is being retained. SDS-PAGE allows a visual evaluation of the purification scheme
Specific activity
The ratio of enzyme activity to total protein concentration. Specific activity should increase with each step of the purification procedure since total protein is being removed while desired enzyme is being retained
Zonal (gradient) centrifugation
Used to separate proteins with different sedimentation coefficients. A density gradient is formed in a centrifuge tube, using differing proportions of a low density solution and a high density solution, like 5% and 20% sucrose. The solutions are mixed and create a linear gradient of sucrose concentration, which is highest at the bottom of the tube and lowest at the top. It stabilizes the sedimentation of the molecules. On centrifugation, particles move away from the starting zone with velocities determined both by their size and shape and by the centrifugal force to which they are subjected. After centrifugation for a certain time, particles will be found in a series of zones spaced according to the relative velocities of the particles. In this way particles differing in sedimentation rate by 20% or less can be separated without undue difficulty. Rate-zonal centrifugation thus complements differential centrifugation. The separated bands (zones) of proteins can be harvested by making a hole in the bottom of the tube and collecting drops
Sedimentation coefficients
Usually expressed in Svedberg units (S). The smaller the S value, the more slowly a molecule moves in a centrifugal field
What are the protein drops collected during zonal centrifugation analyzed for?
Protein content, catalytic activity, other biochemical characteristics or functional properties
Advantages of recombinant technology (3)
- Proteins can be expressed in large quantities
- Affinity tags can be fused to proteins that allow
purification of the protein or visualization of the protein in the cell - Proteins with modified primary structures can be readily generated
Antibody
Also called an immunoglobulin. A protein synthesized by vertebrates in response to the presence of an antigen. Antibodies are specific and have a high affinity for the antigens that elicited their synthesis. The binding of an antibody to an antigen is a step in the immune response that protects the animal from infection
Antigen
A foreign substance, antibodies are synthesized in response to an antigen. Foreign proteins, polysaccharides, and nucleic acids can be antigens. Small foreign molecules like synthetic peptides can also act as antigens if the small molecule is attached to a macromolecular carrier
Antigenic determinant (epitope)
A specific group or cluster of amino acids on the target molecule that can be recognized by an antibody
Antibody structure
Antibodies can contain multiple chains, linked by disulfide bonds. One antibody can have multiple domains and multiple antigen binding sites. For example, IgG has 3 domains (2 Fab domains and 1 Fc domain). There is an antigen binding site on each Fab domain
Antibody-antigen interactions IgG
A protein antigen binds to the end of an Fab domain of an antibody. The end of the antibody and the antigen have complementary shapes, which allows a large amount of surface to buried when the two proteins bind
Polyclonal antibodies
Antibodies derived from multiple antibody-producing cell populations. Each recognizes a different surface feature of the same antigen. They are advantageous for the detection of a protein of low abundance, because each protein molecule can be bound by more than one antibody at multiple distinct antigenic sites
Monoclonal antibodies
Identical antibodies that
recognize only one epitope- synthesized by clones of a single antibody producing cell
Monoclonal antibody producing cell lines
Immortal cell lines that produce monoclonal antibodies are derived from multiple myeloma (cancer of antibody producing cells). A cancerous plasma cell can generate a very large number of the same kind of cell. These cells are clones because they are descended from the same cell and have identical properties. The identical cells secrete large amounts of a single immunoglobulin
Hybridoma cells
Large amounts of antibodies of any desired specificity can be obtained by fusing a short lived antibody producing cell with an immortal myeloma cell. An antigen is injected into a mouse, and its spleen is removed after several weeks. A mixture of plasma cells from the spleen are fused in vitro with myeloma cells. Each of the resulting hybrid cells are called hybridoma cells, and they indefinitely produce the identical antibody specified by the parent cell from the spleen.
Preparation of monoclonal antibodies
The hybrid cells are allowed to proliferate by growing them in selective medium. Hybridoma cells can be screened by a specific assay for the antigen-antibody interaction to determine which ones produce antibodies of the preferred specificity.
Enzyme-linked immunosorbent assay (ELISA)
Makes use of an enzyme that reacts with a colorless substrate to produce a colored product. The enzyme is covalently linked to a specific antibody that recognizes a target antigen. If the antigen is present, the antibody-enzyme complex will bind to it and on addition of the substrate, the enzyme will catalyze the reaction, generating the colored product. Therefore, the presence of the colored product indicates the presence of the antigen.
Indirect ELISA
Used to detect the presence of an antibody and is the basis of the test for HIV infection. In an indirect ELISA, the production of color indicates the amount of an antibody to a specific antigen
How does the test for HIV infection work?
The test for HIV infection is a quantitative assay- the rate of the color formation reaction is proportional to the amount of antibody originally present. It is carried out through indirect ELISA. The test detects the presence of antibodies that recognize viral core protein antigens. Viral core proteins are adsorbed to the bottom of a well. Antibodies from the person being tested are then added to the coated well. Only someone infected with HIV will have antibodies that bind to the antigen. Enzyme-linked antibodies to human antibodies are allowed to react in the well, and unbound antibodies are removed by washing. An enzyme reaction yielding a colored product suggests the enzyme linked antibodies are bound to human antibodies, and the person is positive for HIV
Steps of indirect ELISA (4)
- A well is coated with the target antigen (like viral core proteins)
- Specific antibody binds to antigen (human antibodies from the sample)
- Enzyme linked antibody binds to specific antibody (antibodies that recognize human antibodies)
- Substrate is added and converted by enzyme into colored product, the rate of color formation is proportional to the amount of specific antibody
Adsorb
Hold (molecules of a gas or liquid or solute) as a thin film on the outside surface or on internal surfaces within the material.
Sandwich ELISA steps (4)
- A well is coated with monoclonal antibodies
- Antigen binds to antibody
- A second monoclonal antibody, linked to the enzyme, binds to the immobilized antigen
- Substrate is added and converted by the enzyme into a colored product. The rate of color formation is proportional to the amount of antigen
Sandwich ELISA
Used to detect antigens instead of antibodies. The antibody is a specific antigen is adsorbed to the bottom a well. The solution containing the antigen (like blood or urine) is added to the well and binds to the antibody. A second antibody that recognizes the antigen is added. This antibody is enzyme linked
Western blotting
An immunoassay technique used to detect very small quantities of a protein of interest in a cell or body fluid. A sample undergoes SDS-PAGE. A polymer sheet is pressed against the gel, transferring the resolved proteins on the gel to the sheet, which makes the proteins more accessible for reaction. The primary antibody is added to the sheet and reacts with the antigen. The antibody-antigen complex on the sheet can be detected by rinsing the sheet with a secondary antibody. The secondary antibody is generally fused to an enzyme that produces a colored product or is fluorescently labeled. Western blotting makes it possible to find a protein in a complex mixture, the proverbial needle in a haystack.
Primary antibody
In western blotting, an antibody specific for the protein of interest
Secondary antibody
In western blotting, an antibody specific for the primary antibody
What diseases is western blotting used to test for?
It tests for infection by hepatitis C- it can detect a core protein of the virus. It can also be used to monitor protein purification and for the cloning of genes
Co-immunoprecipitation
Uses a monoclonal antibody against a specific protein to determine the binding partners of that protein in vivo. The sample of interest is incubated with the specific antibody. Agarose beads coated with an antibody binding protein are added to the mixture. The antibody binding protein recognizes a portion of the antibody that is separate from the antigen binding region, and therefore does not disrupt the protein-antibody complex. After centrifugation, the antibody, which is now bound to the beads, aggregates at the bottom of the tube. The antibody-protein complex will also precipitate any additional proteins bound to the original protein. The precipitate can be analyzed with SDS-PAGE and western blotting to identify the binding partners
Precipitate definition
Cause (a substance) to be deposited in solid form from a solution.
Fluorescence microscopy
Cells are stained with fluorescence labeled antibodies and examined under a microscopy to determine the location of a protein of interest
Green fluorescent protein
A naturally fluorescent protein isolated from the jellyfish Aequorea Victoria
Mass spectrometry
Allows the highly precise and sensitive measurement of the atomic composition of a specific molecule without prior knowledge of its identity. You can use this method to determine the mass of an analyte. This information can be used to determine the identity and chemical state of the molecule of interest
How do mass spectrometers work?
They convert analyte molecules into gaseous, charged forms (gas phase ions). Electrostatic potentials are applied to measure the ratio of the mass of each ion to its charge (mass to charge ratio)
Matrix-assisted laser desorption/ionization (MALDI)
The molecule is evaporated to dryness in the presence of a volatile, aromatic compound (the matrix) that can absorb light at specific wavelengths. A laser pulse tuned to one of these wavelengths excites and vaporizes the matrix, converting some of the analyte into the gas phase. It converts the analyte into gas phase ions (ionization) which is necessary for mass spectrometry
Time of flight (TOF) mass analyzer
Ions are accelerated through an elongates chamber under a fixed electrostatic potential. A smaller ion will require less time to travel across the chamber than the larger ion will. The mass of each ion can be be determined by measuring the time required for each ion to pass through the chamber
3 components of mass spectrometers
An ion source, a mass analyzer, and a detector
Mass spectrum
The mass spectrometer detects ions on the basis of their mass to charge ratio. The ions will appear as separate peaks in the mass spectrum
Edman degradation
A chemical method of peptide sequencing. The N-terminal amino acid of a polypeptide is labeled with phenyl isothiocyanate. Subsequent cleavage creates the PTH-amino acid derivative and the polypeptide chain, which has been shortened by one residue. The PTH amino acid can be identified by spectroscopic methods (like high performance liquid chromatography). The procedure can be repeated on the shortened peptide, which creates another PTH amino acid. The procedure is repeated until you get the full sequence
Tandem mass spectrometry
The use of mass spectrometry for protein sequencing takes advantage of the fact that ions of proteins have been analyzed by a mass spectrometer (precursor ions) can be broken into smaller peptide chains by bombardment with atoms of an inert gas like helium or argon. These new fragments (product ions) can be passed through a second mass analyzer for further mass characterization. The product-ion fragments are formed in chemically predictable ways that can provide clues to the amino acid sequence of the precursor ion
Why is Edman degradation limited to peptides of 50 residues?
Not all peptides in the reaction mixture release the amino acid derivative at each step, creating an impure mix. Also, sequencing of long peptides by mass spectrometry creates a mass spectrum that can be complex and difficult to interpret. This can be resolved by cleaving a protein into smaller peptides that can be sequenced. Protein cleavage can be achieved by chemical reagents of proteolytic enzymes
Trypsin
Cleaves the carboxyl side of lysine and arginine residues
Clostripain
Cleaves the carboxyl side of arginine residues
Staphylococcal protease
Cleaves the carboxyl side of aspartate and glutamate residues (glutamate only under certain conditions)
Thrombin
Cleaves the carboxyl side of arginine
Chymotrypsin
Cleaves the carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine
Carboxypeptidase A
Cleaves the amino side of C-terminal amino acid (not arginine, lysine, or proline)
Overlap peptides
A second enzyme is used to split the polypeptide chain at different linkages. For example, chymotrypsin cleaves preferentially on the carboxyl side of aromatic and some other bulky nonpolar residues. Because these chymotryptic peptides overlap two or more tryptic peptides, they can be used to establish the order of the peptides. The entire amino acid sequence of the polypeptide chain is then known
Disulfide bond reduction
Polypeptides linked by disulfide bonds can be separated by reduction with dithiothreitol followed by alkylation of the cysteine residues to prevent them from re-forming. This is done to separate polypeptide chains for sequencing
Other than overlapping peptides, which steps are necessary if the protein sample is several polypeptide chains?
Denaturing agents (urea and guanidine hydrochloride) are used to dissociate chains held together by noncovalent bonds. Disulfide bonds are also dissociated. SDS-PAGE under reducing conditions should display the number of chains
Why are genomic and proteomic methods considered complementary?
Genomic techniques that reveal the DNA sequence of
the gene encoding a protein also allow determination of
the amino acid sequence. Recombinant DNA technology has generated a lot of amino acid sequence information. Although we are able to determine the sequence of the nascent amino acid, we are unable to determine the sequence after posttranslational modifications
Information provided by the amino acid sequence (6)
- We can compare the sequence of a protein of interest to other known sequences to categorize the protein and gain information about the protein’s structure and function
- Provides information about evolutionary pathways and genealogical relationships between species
- The sequence can contain internal repeats that reveal the history of the individual protein
- Some amino acid sequences serve as signals designating their destinations or controlling their processing
- Sequence data provides a basis for preparing antibodies specific for a protein of interest
- Amino acid sequences can be used to make DNA probes that are specific for the genes encoding the corresponding proteins
Uses of synthetic peptides (4)
- Serve as an antigen to stimulate the formation of specific antibodies
- Isolate receptors for many hormones and other signal molecules
- Serve as drugs- vasopressin is an example
- Define the rules governing the 3D structure of proteins. Does a specific sequence form a certain shape by itself?
Vasopressin
Also called antidiuretic hormone. A synthetic peptide hormone that stimulates the reabsorption of water in the distal tubules of the kidney (the collecting ducts). This causes the formation of more concentrated urine. Patients with diabetes are deficient in ADH and excrete large volumes of dilute urine
Formylmethionyl (fMet) peptides
White blood cells are attracted to bacteria by fMet peptides released in the breakdown of bacterial proteins. Synthetic fMet peptides have been used to identify the cell surface receptor for this class of peptides. Synthetic peptides can also be attached to agarose beads for affinity chromatography columns for the purification of receptor proteins that specifically recognize the peptides
How are synthetic peptides constructed?
The amino group of one amino acid is linked to a carboxyl group of another. A unique product will only be formed if a single amino group and a single carboxyl group are available for reaction. Therefore, you need to block some groups and activate others to prevent unwanted reactions. The carboxyl terminal amino acid is attached to an insoluble resin by its carboxyl group, which prevents it from participating in more peptide bond forming reactions. The alpha amino group of this amino acid is blocked with a protecting group like t-Boc. t-Boc is then removed with trifluoroacetic acid
Steps of solid phase peptide synthesis (4)
- Anchoring the C-terminal amino acid to a solid resin
- Deprotection of the amino terminus
- Coupling of the free amino terminus with the DCC activated carboxyl group of the next amino acid
Steps 2 and 3 are repeated for each added amino acid. - The completed peptide is released from the resin
Why is it important to determine 3D protein structure?
The 3D structure of a protein gives information about the protein’s function. The specificity of active sites and binding sites is defined by the precise atomic arrangement within these regions
X-Ray crystallography
Used to determine protein structure in atomic detail. You can view the precise 3D positions of most atoms in a protein. An X-ray source generates a beam, which is diffracted by a crystal. The resulting diffraction pattern is collected on a detector
Why do X-Rays provide the best resolution for determining molecular structures?
Their wavelength approximately corresponds to the length of a covalent bond
3 components in X-Ray crystallographic analysis
A protein crystal, a source of x-rays, and a detector
Preparation of proteins for X-ray crystallography
A protein has to be prepared in crystal form, so all protein molecules are oriented in a fixed, repeated arrangement with respect to one another. Adding ammonium sulfate or another salt to a concentrated solution of proteins favors the formation of highly ordered crystals- this is the process of salting out
Basic physical principles underlying X-ray crystallography (3)
- Electrons scatter X-rays. The amplitude of the wave scattered by an atom is proportional to its number of electrons
- The scattered waves recombine. each diffracted beam comprises waves scattered by each atom in the crystal
- The way in which the scattered waves recombine depends on the atomic arrangement.
When do waves reinforce one another or cancel each other out?
The waves reinforce each other if they are in phase. They cancel one another if they are out of phase
Reflections
The protein crystal is rotated so that the beam can strike the crystal from many directions. This rotational motion makes an x-ray photograph that is made up of a regular array of spots called reflections. The intensities and positions of these reflections are the basic experimental data of an x-ray crystallographic analysis. An x-ray diffraction pattern is produced
Nuclear magnetic resonance (NMR) spectroscopy
Reveals the atomic structure of macromolecules in solution. based on the fact that certain atomic nuclei are intrinsically magnetic and can exist in two spin states when an external magnetic field is applied
Principle behind NMR
Certain atomic nuclei have magnetic properties, called spin. The hydrogen nucleus (a proton) is an example of an isotope that has this property. The spinning of a proton generates a magnetic moment. The moment can take either an alpha or beta spin state as its orientation when an external magnetic field is applied. The alpha state has a slightly lower energy because it is aligned with this applied field. A spinning proton in an alpha state can be raised to an excited beta state when exposed to electromagnetic radiation that corresponds in frequency to the difference between the alpha and beta states. The spin will change from alpha to beta and resonance will be obtained
Shielding
The small, local magnetic field that opposes the applied field. Shielding is produced by the flow of electrons around the magnetic nucleus. The degree of shielding depends on the surrounding electron density. Therefore, nuclei in different environments will resonate (change states) at slightly different radiation frequencies. The chemical shift will be lower if the nucleus is shielded.
Chemical shifts
The different frequencies of electromagnetic radiation absorbed by the nuclei of the sample. The chemical shifts depend on the environment of the nuclei,
and the environment depends on protein structure
One dimensional NMR spectra
Reveals changes to a particular chemical group under different conditions. One dimensional NMR spectra show the peaks of groups at different chemical shifts depending on the type of group and its environment
Two dimensional NMR spectra (NOESY)
Displays groups that are in close proximity, even if they are not close together in the primary structure. The Nuclear Overhauser effect is the basis of this technique
Nuclear Overhauser effect
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 the two nuclei are less than 5A apart. The effect provides a means of detecting the location of atoms relative to one another in the 3D structure of the protein. It identifies pairs of protons that are in close proximity
NOESY spectrum
Shows the peaks of 2D NMR. The 3D structure of a protein can be reconstructed with the use of the proximity relations from the spectrum. Proton pairs identified by NOESY must be separated by less than 5A in the 3D structure. If enough distance constraints are applied the unique 3D structure can be accurately determined
Cryo-electron microscopy
A thin layer of the protein solution is prepared in a fine grid then frozen very quickly, which traps the molecules in a variety of orientations. The sample is placed in a transmission electron microscope under vacuum conditions and exposed to an incident electron beam. Each protein interacts with the beam to produce a 2D projection on the detector. Many projections are detected, and each capture a molecule in a different orientation
Single particle analysis
The process by which computers use the projections from cryo-electron microscopy to build a 3D representation of the protein
When is cryo-electron microscopy used?
Certain proteins are difficult to visualize. Proteins that form large macromolecular complexes or are embedded in the lipid membrane can be viewed by cryo-electron microscopy. It was used to identify binding sites for TRPV1, a tetrameric transmembrane protein
5 immunoglobulins
IgG, IgD, IgA, IgE, IgM
Parathyroid gland
Produces parathyroid hormone, which plays a key role in the regulation of calcium levels in the body. Small changes to calcium can cause muscle and nerve problems
Functions of the parathyroid gland (4)
- Release of calcium by bones into the bloodstream
- Absorption of calcium from food by the intestines
- Conservation of calcium by the kidneys
- Stimulates cells in the kidney to transforms weaker forms of vitamin D into the form that is strongest at absorbing calcium from the intestines
Weight limit of NMR
Proteins should be less than 50 kDa
The speed at which particles sediment depends on
Size, density, and mass- also depends on the properties of the fluid
