Biochem Module 3

What is required to purify a protein?

An assay that determines whether the protein of interest is present.

What does an assay for lactate dehydrogenase detect?

NADH, spectrophotometrically.

What is specific activity?

The ratio of enzyme activity to the amount of total protein in a mixture.

What is the overall goal of protein purification, in terms of specific activity?

To maximize the specific activity.

What is a cell homogenate?

The product of disrupting cell membranes of intact cells.

What is differential centrifugation?

Repeated centrifugation at increasing speeds to yield fractions of decreasing density.

What is "salting out"?

The effect by which most proteins become less soluble at high salt concentrations.

What is dialysis used for in protein purification?

Separating proteins from small molecules (like salt) using a semipermeable membrane.

In dialysis, what happens to molecules smaller than the membrane pore size?

They diffuse out of the dialysis bag down their concentration gradient.

What does gel-filtration (molecular exclusion) chromatography separate proteins by?

Size.

In gel-filtration chromatography, which proteins exit the column first?

Large proteins, because they cannot enter the porous beads.

What does ion-exchange chromatography separate proteins by?

Charge.

How are bound proteins released from an ion-exchange column?

By increasing the salt concentration of the buffer.

What type of beads does cation exchange chromatography use?

Negatively-charged beads.

What type of beads does anion exchange chromatography use?

Positively-charged beads.

What does affinity chromatography exploit to separate proteins?

A protein's high affinity for a specific molecule called a ligand.

How is a bound protein released from an affinity chromatography column?

By passing a solution enriched in the ligand through the column.

What is HPLC (high-performance liquid chromatography)?

A chromatography technique using very fine beads and pressure for sharper, more rapid protein separations.

Why do very fine beads increase resolving power in chromatography?

They allow more potential sites of interaction between the protein and the beads.

What is electrophoresis?

A technique that separates charged molecules by applying an electric field, often through a gel.

What does SDS do in SDS-PAGE?

SDS is an anionic detergent that denatures proteins and gives them a uniform charge-to-mass ratio.

How many SDS molecules typically bind per amino acid in a protein?

Approximately 1 molecule of SDS per 2 amino acids.

What do proteins separated by SDS-PAGE migrate based on?

Mass only, since SDS gives them equal charge-to-mass ratios.

What dye is commonly used to stain proteins after SDS-PAGE?

Coomassie blue.

How does electrophoretic mobility relate to protein mass in SDS-PAGE?

It is linearly proportional to the logarithm of the protein's mass.

What is the isoelectric point (pI) of a protein?

The pH at which the protein has no net charge.

What is isoelectric focusing?

A technique that separates proteins in a pH gradient gel based on their isoelectric point (pI).

What is a protein's electrophoretic mobility at its pI?

Zero.

What two techniques are combined in two-dimensional electrophoresis?

Isoelectric focusing (horizontal) and SDS-PAGE (vertical).

What two properties does two-dimensional electrophoresis separate proteins by?

pI (horizontal direction) and mass (vertical direction).

How should specific activity change throughout a successful purification scheme?

It should increase at each step, as total protein decreases while the desired enzyme is retained.

What does ultracentrifugation help analyze about biomolecules?

Mass, density, shape, and interactions with other molecules.

What are sedimentation coefficients measured in?

Svedberg units (S).

How does particle mass relate to sedimentation rate?

A more massive particle sediments more rapidly than a less massive one.

How does particle shape affect sedimentation rate?

Elongated particles sediment more slowly than spherical particles of the same mass.

What advantages does recombinant DNA technology offer for protein purification?

Large-scale expression, addition of affinity tags, and generation of proteins with modified primary structure.

What is an antibody?

A protein synthesized in response to a foreign substance called an antigen.

What is an epitope (antigenic determinant)?

The specific group or cluster of amino acids on a target molecule that an antibody recognizes.

What are polyclonal antibodies?

A heterogeneous mixture of antibodies from multiple cell populations, each specific for a different epitope.

What are monoclonal antibodies?

Identical antibodies produced by clones of a single antibody-producing cell.

What is a hybridoma cell?

A hybrid cell formed by fusing an antibody-producing cell with an immortal myeloma cancer cell.

What is ELISA used for?

Quantifying the amount of a specific protein or antigen using an antibody linked to an enzyme.

What enzyme is commonly linked to antibodies in ELISA?

Horseradish peroxidase.

What is a primary antibody in western blotting?

An antibody specific for the target protein of interest.

What is a secondary antibody in western blotting?

An antibody specific for the primary antibody, often linked to a detection enzyme or fluorescent tag.

What is western blotting used for?

Detecting proteins that have been separated by gel electrophoresis using antibody staining.

What is co-immunoprecipitation used to identify?

Binding partners of a specific protein.

What agarose bead coating is commonly used in co-immunoprecipitation?

Beads coated with an antibody-binding protein, such as Protein A.

What can fluorescence-labeled antibodies reveal about a protein?

Its cellular location, via fluorescence microscopy.

What is immunoelectron microscopy used for?

Visualizing proteins with very fine spatial resolution using antibodies labeled with electron-dense metal.

What does mass spectrometry measure?

The mass-to-charge ratio (m/z) of gas-phase ions from an analyte.

What are the three main components of a mass spectrometer?

An ion source, a mass analyzer, and a detector.

What is MALDI?

Matrix-assisted laser desorption/ionization, a technique where the analyte is evaporated with a light-absorbing matrix.

What is ESI (electrospray ionization)?

A technique where a solution of the analyte is passed through an electrically charged nozzle to form ions.

How does a time-of-flight (TOF) mass analyzer determine ion mass?

By measuring the time required for each accelerated ion to pass through a chamber.

What is Edman degradation used for?

Sequencing peptides by sequentially removing and identifying the N-terminal amino acid.

What reagent reacts with the N-terminal amino acid in Edman degradation?

Phenyl isothiocyanate (PTH).

What has largely replaced Edman degradation for protein sequencing?

Mass spectrometry (particularly tandem mass spectrometry).

What happens in tandem mass spectrometry?

Precursor ions are broken into smaller fragments, and product ions are passed through a second mass analyzer.

What is the peptide length limitation of Edman degradation?

It is limited to peptides of about 50 residues or fewer.

Why must proteins be cleaved into smaller peptides before full sequencing?

Because Edman degradation and mass spectrometry work best on peptides under 50 residues.

How are peptide fragments ordered to reconstruct a full protein sequence?

By using a different cleavage procedure to generate overlapping peptides.

Where does cyanogen bromide cleave a polypeptide?

On the carboxyl side of methionine residues.

Where does trypsin cleave a polypeptide?

On the carboxyl side of lysine and arginine residues.

Where does chymotrypsin cleave a polypeptide?

On the carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine.

Where does hydroxylamine cleave a polypeptide?

At asparagine-glycine bonds.

How are disulfide bonds cleaved for protein sequencing?

By reduction with thiols such as β-mercaptoethanol or dithiothreitol.

How is disulfide bond reformation prevented after reduction?

By alkylating the cysteine residues with iodoacetate.

Why is recombinant DNA technology sometimes preferred for sequencing large proteins?

It can be more efficient than direct protein sequencing methods for very large proteins.

What is peptide mass fingerprinting?

A technique for identifying an individual protein via specific cleavage, chromatography, and mass spectrometry.

What is the proteome?

The entire set of proteins expressed and modified by a cell under a particular set of conditions.

How does the proteome differ from the genome?

The proteome is not fixed and changes with cellular conditions, unlike the genome.

What are three uses of synthetic peptides?

Serving as antigens, isolating receptors, and serving as drugs.

In solid-phase peptide synthesis, what is the growing chain attached to?

An inert matrix, via its carboxyl terminus.

What protecting groups are used to block the α-amino group in solid-phase synthesis?

tert-Butyloxycarbonyl (t-Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc).

What removes the protecting group in solid-phase peptide synthesis?

Trifluoroacetic acid.

What is the role of dicyclohexylcarbodiimide (DCC) in solid-phase synthesis?

It activates the carboxyl group of the incoming amino acid for peptide bond formation.

What does x-ray crystallography reveal about proteins?

Their three-dimensional structure in atomic detail.

What underlying physical process makes x-ray crystallography possible?

Electrons of atoms scatter x-rays, and the scattered waves recombine based on atomic arrangement.

What mathematical operation converts an x-ray diffraction pattern into an electron density map?

A Fourier transform.

What physical property of atomic nuclei does NMR spectroscopy exploit?

Certain atomic nuclei are intrinsically magnetic and can exist in two spin states in a magnetic field.

What are "chemical shifts" in NMR spectroscopy?

The different frequencies at which nuclei absorb electromagnetic radiation, dependent on their environment.

What is examined in two-dimensional NMR?

How altering the spin of one nucleus affects the spin of a neighboring nucleus.

What is the Nuclear Overhauser Effect (NOE) used to identify?

Pairs of protons that are in close physical proximity.

What is cryo-electron microscopy (cryo-EM) used for?

Determining the structures of large proteins and macromolecular complexes.

What happens to a protein sample in cryo-EM preparation?

It is frozen in a thin layer, trapping molecules in an ensemble of orientations.

How does cryo-EM generate a 3D protein structure?

A computer combines multiple 2D projections obtained via transmission electron microscopy into a 3D representation.