Molecular Biology Chapter 2

isoelectric pH (pI)

(pI, pH(I), IEP), is the pH at which a particular molecule carries no net electrical charge.

dalton

1 atomic mass unit (1/12 of a carbon atom)

kilodalton (kD or kDa)

1000 daltons

beta-mercaptoethanol (2-ME)

2-Mercaptoethanol is the chemical compound with theformula HOCH2CH2SH. ME or ßME, as it is commonly abbreviated, and is used to reduce disulfide bonds

phosphorylation

A biochemical process that involves the addition of phosphate to an organic compound. Phosphorylation is carried out through the action of enzymes known as phosphotransferases or kinases.

molecular or gel exclusion (size) chromatography

A chromatographic method in which molecules in solution are separated by their size, and in some cases molecular

ion exchange (charge) chromatography

A chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids. The ion exchanger could be beads with negative (anion) or positive (cation) charge.

domain

A conserved part of a given protein sequence and (tertiary) structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded.

polypeptide

A linear organic polymer consisting of a large number of amino-acid residues bonded together in a chain, forming part of (or the whole of) a protein molecule

affinity chromatography

A method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.

ligand

A molecule that binds to another (usually larger) molecule.

pH

A numeric scale used to specify the acidity or basicity of an aqueous solution. It is approximately the negative of the base 10 logarithm of the molar concentration, measured in units of moles per liter, of hydrogen ions. More precisely it is the negative of the logarithm to base 10 of the activity of the hydrogen ion. Solutions with a pH less than 7 are acidic and solutions with a pH greater than 7 are basic.

fraction

A portion or part of the whole. Depending on the molecular biology context, a fraction may be representative of the whole, or, a fraction could be a separated portion. In chromatography, a fraction is a separated portion of the whole that is not representative of the whole.

homogenization

A process whereby a biological sample is brought to a state such that all fractions of the sample are equal in composition. A homogenized sample called the homogenate and is equal in composition throughout. If a portion (aliquot) is removed, removal of the aliquot does not alter the overall molecular make-up of the sample remaining, and is identical to the aliquot removed.

electrophoresis

A process which enables the sorting of molecules based on size. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar or polyacrylamide. The electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel

binding site

A region on a biological macromolecule (protein, nucleic acid, carbohydrate, or lipid) to which ligands (specific molecules and/or ions) may form a chemical bond. An equilibrium exists between unbound ligands and bound ligands.

chromatography

A set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases

leucine zipper motif

A structural motif found in a number of proteins (some of the DNA-binding regulatory proteins) in which leucyl residues align along one edge of the helix and can interdigitate with a similar structure on another protein molecule

coiled-coil motif

A structural motif in proteins in which 2-7[1] alpha-helices are coiled together like the strands of a rope (dimers and trimers are the most common types). Many coiled coil-type proteins are involved in important biological functions such as the regulation of gene expression, e.g. transcription factors.

solute

A substance that is dissolved in another substance (a solvent), forming a solution.

cell extract

After breaking open cells, a preparation of all cell constituents or a subset of cellular material.

hydrophobic residue

An amino acid with an R-group that is hydrophobic

kinase

An enzyme that catalyzes the transfer of the gamma phosphate group from ATP to a specified molecule.

dimer

An oligomer consisting of two structurally similar monomers joined by bonds that can be either strong or weak, covalent or intermolecular. The term homodimer is used when the two molecules are identical (e.g. A-A) and heterodimer when they are not (e.g. A-B).

peak [as used in chromatography]

As the separated molecules leave chromatography, the molecules pass immediately into a detector. The detector sees [detects] each separated compound band An appropriate detector [UV, ELS, fluorescence, mass...etc.] has the ability to sense the presence of a compound and send its corresponding electrical signal to a computer data station where it is recorded as a peak. The detector responds to the varying concentration of the specific molecules within the band, where the center of the band [highest population of analyte molecules] is interpreted by the detector as the apex of the peak.

anode and cathode

As used in electrophoresis, moledules that are positively charged will migrate towards the cathode which is negatively charged (because this is an electrolytic rather than galvanic cell). If the species are negatively charged they will migrate towards the positively charged anode

motif

In a chain-like biological molecule, such as a protein or nucleic acid, it is a supersecondary structure, which also appears in a variety of other molecules. do not allow us to predict the biological functions: they are found in proteins and enzymes with dissimilar functions. are between domains and secondary structure in complexity.

phi (ö) angle

In peptides, the angle of the bond between the alpha carbon and the amine group

psi (ø) angle

In peptides, the angle of the bond between the alpha carbon and the carboxyl group

chaperonin

Newly made proteins usually must fold from a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, called molecular chaperones. The potential energy to assist in protein folding is supplied by structural changes imposed by adenosine triphosphate (ATP) binding and/or hydrolysis.

polar residue

Polar amino acid

SDS-PAGE

Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule. As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure, or a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured linear chain whose mobility depends only on its length and mass-to-charge ratio. For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to linearize proteins and to impart a negative charge to linearized proteins. This procedure is called SDS-PAGE. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for proteins or urea for nucleic acids. SDS is an anionic detergent that denatures secondary and non-disulfide-linked tertiary structures, and additionally applies a negative charge to each protein in proportion to its mass. Urea breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to separate. Heating the samples to at least 60 °C further promotes denaturation. In addition to SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits).

lane [as used in electrophoresis]

Protein and nucleic acid samples are added to sample wells at the top of the gel. When the electrical current is applied, the molecules move down through the gel matrix, creating what are called "lanes" of protein or nucleic acid "bands". Samples that are loaded in adjacent wells and electrophoresed together are easily compared to each other after staining or other detection strategies. The intensity of staining and "thickness" (not location) of bands are indicative of their relative abundance. The positions (locations) of bands within their respective lanes indicate their relative sizes (and/or other factors affecting their rate of migration through the gel).

denatured

Proteins or nucleic acids that have lost their quaternary structure, tertiary structure and secondary structure which is present in their native state. In vitro, molecules can be denatured by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), radiation or heat. If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Protein denaturation is also a consequence of cell death. Denatured proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility to aggregation due to the exposure of hydrophobic groups.

polypeptide carboxy (C or COOH) terminus

The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is translated from messenger RNA, it is created from N-terminus to C-terminus. The convention for writing peptide sequences is to put the C-terminal end on the right and write the sequence from N- to C-terminus.

polypeptide amino (N or NH2) terminus

The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide terminated by an amino acid with a free amine group (-NH2).

alpha carbon

The carbon atom next to the carboxyl group (which is therefore numbered 2 in the carbon chain starting from that functional group) is called the a-carbon. Amino acids containing an amino group bonded directly to the alpha carbon are referred to as alpha amino acids

Anfinsen experiment

The hypothesis that "protein amino acid sequence determines the final shape a protein assumes in a water solution" was proven to be correct when Christian B. Anfinsen showed that if the enzyme ribonuclease was opened out into a linear chain and then allowed to reform, it reassumed the correct catalytic shape

ionization

The process by which an atom or a molecule acquires a negative or positive charge by gaining or losing electrons to form ions, often in conjunction with other chemical changes.

hydrophobic interaction

The tendency of nonpolar molecules in a polar solvent (usually water) to interact with one another. The interactions between the nonpolar molecules are called hydrophobic interactions.

peptide bond

a chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the other molecule, releasing a molecule of water (H2O). This is a dehydration synthesis reaction (also known as a condensation reaction), and usually occurs between amino acids.

alpha helix

a common motif in the secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid located three or four residues earlier along the protein sequence.

aromatic base

a cyclic (ring-shaped), planar (flat) molecule with a ring of resonance bonds that exhibits more stability than other geometric or connective arrangements with the same set of atoms.

coomasie blue protein stain

a family of dyes commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent ("destaining").

ATP binding site

a protein micro-environment where ATP is captured and hydrolyzed to ADP, releasing energy that is utilized by the protein to "do work" by changing the protein shape and/or making the enzyme catalytically active.

disulfide bond

also called an S-S bond, or disulfide bridge, is a covalent bond derived from two thiol groups. The most common way of creating this bond is by the oxidation of sulfhydryl groups.

amphipathic helix

an alpha helix with opposing polar and nonpolar faces oriented along the long axis of the helix.

reducing agent

an element compound (e.g. 2ME) that loses (or "donates") an electron to another chemical species in a redox chemical reaction. losing electrons, it is said to have been oxidized.

van der Waals interactions

are the residual attractive or repulsive forces between molecules or atomic groups that do not arise from covalent bonds, nor ionic bonds. can be attractive or repulsive. They are caused by correlations in the fluctuating polarizations of nearby particles (a consequence of quantum dynamics).

helix-turn-helix motif

found in DNA binding proteins that regulate gene expression. The motif consists of approximately 20 residues and is characterized by 2 alpha-helices, which make intimate contacts with the DNA and the helices are joined by a short sequence of amino acids that make the turn. The second helix of the HTH motif binds to DNA via a number of hydrogen bonds and hydrophobic interactions, which occur between specific side chains and the exposed bases and thymine methyl groups within the major groove of the DNA

protein quaternary structure

involves the clustering of two or more polypeptides (they may or may not be the same) into a final specific shape. Each of the proteins is called a subunit. In the context of proteins that have quaternary structure, the final structure itself is called a protein - a protein with subunits. Each of the individual subuntis are themselves, a protein. A variety of bonding interactions including hydrogen bonding, salt bridges, and disulfide bonds hold the various chains into a particular geometry. There are two major categories of proteins with quaternary structure - fibrous and globular. The multi

amino acid R-group (side chain)

organic compounds which contain both an amino group and a carboxyl group. They are distinguished by the attached functional group R. ... The simplest of the amino acids, glycine, has just a hydrogen atom in the position of the R-group.

well [as used in electrophoresis]

small indentations created in the gel when it is made. uniformly spaced along the side of the gel closest to the negative electrode. The even, linear spacing of the wells provides a uniform starting position for the samples. also allow the samples to be placed into the gel so that when current is applied, the samples are pulled through the middle of the gel, not across the top.

molecular weight

synonymous with molecular mass. Ratio of the mass of a molecule to the atomic mass unit. (It is more appropriate to use molecular mass instead of molecular weight, but in Molecular Biology, both molecular weight and molecular mass are used as synonyms.)

ionic bond

the complete transfer of valence electron(s) between atoms. It is a type of chemical bond that generates two oppositely charged ions.

band

the detected molecules. If separating by size, then a band is the detected molecules of a distinct size. If separating by charge, then a band is molecules of a certain charge. In SDS-PAGE of proteins from a cell, many bands are since there are many proteins of different sizes. A band in SDS-PAGE contains thousands of proteins of the same size. In DNA gel electrophoresis, a band contains thousands of DNA molecules of the same size.

hydrogen bond

the electrostatic attraction between two polar groups that occurs when a hydrogen (H) atom covalently bound to a highly electronegative atom such as nitrogen (N), oxygen (O), or fluorine (F) experiences the electrostatic field of another highly electronegative atom nearby. Hydrogen bonds can occur between molecules (intermolecular) or within different parts of a single molecule (intramolecular)

protein tertiary structure

the final specific geometric shape that a protein assumes. This final shape is determined by a variety of bonding interactions between the "side chains" on the amino acids. These bonding interactions may be stronger than the hydrogen bonds between amide groups holding the helical structure. As a result, bonding interactions between "side chains" may cause a number of folds, bends, and loops in the protein chain. Different fragments of the same chain may become bonded together. There are four types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions.

renaturation

the reconstruction of a protein or nucleic acid (such as DNA) to their original 3D form especially after denaturation. This process is therefore the inverse of denaturation. Reconstructing the denatured nucleic acid or protein into its original form is done by the process of renaturation. For instance, a heat-denatured DNA can revert to its original form by cooling slowly the two strands which, by chance, finding complementary regions and reform into a double-stranded helix. A denatured protein may be restored following denaturation although it is not as likey to occur as can be done with denatured nucleic acids. One way through which a denatured protein is restored to its original form is by removing the SDS and denaturing agents following denaturation

protein secondary structure

the three dimensional form of local segments of proteins. The two most common secondary structural elements are alpha helices and beta sheets. Secondary structure elements typically spontaneously form as an intermediate before the protein folds into its three dimensional tertiary structureThe secondary protein structure is the specific geometric shape caused by intramolecular and intermolecular hydrogen bonding of amide groups.