Chapter 5 Flashcards
for a protein of n residues, there are … possible sequences
20^n
in general, proteins contain at least … residues or so; polypeptides smaller than that are simply called …
40; peptides
the largest known polypeptide chain belongs to the 35,213 residue …, a giant protein that helps arrange the repeating structures of muscle fibrs
titin
the vast majority of polypeptides contain between … and … residues, with the average being … residues
100; 1000; 355
… proteins contain several identical and/or nonidentical chains called subunits. some proteins are synthesized as single polypeptides that are later cleaved into 2+ chains that remain associated; … is such a protein
multisubunit; insulin
forty residues appears to be near the minimum for a polypeptide chain to … into a discrete and stable shape that allows it to carry out a particular function
fold
polypeptides with well over 1000 residues may approach the limits of …. of the protein synthetic machinery. the longer the polypeptide, the greater the likelihood of introducing … during transcription and translation
efficiency; errors
the most abundant amino acids in proteins are: … … … … … … the rarest amino acids in proteins are: … … … ...
leu ala gly val glu ser
trp
cys
met
his
many proteins consist of more than just amino acid residues. they may form complexes with … such as Zn2+ and Ca2+; they may covalently/noncovalently bind certain small organic molecules; they may be covalently modified by the posttranslational attachment of groups such as … and …
metal ions; phosphates; carbohydrates
biological materials are routinely dissolved in buffer solutions effective in the pH range over which the materials are stable. failure to do so could cause their … (structural disruption), if not their chemical degradation
denaturation
the thermal stability of proteins varies. although some proteins denature at low temps, most proteins denature at high temps, sometimes only a few degrees higher than their native environment. protein purification is normally carried out at temperatures near
0 degrees C
destroying tissues to liberate the molecule of interest also releases degradative enzymes, including … and … these can be inhibited by adjusting the … or … to values that inactivate them or by adding compounds that specifically block their action (provided these methods do not adversely affect the protein of interest)
proteases; nucleases; pH; temperature
many proteins are denatured by contact with the … interface or with … or … surfaces. hence, protein solutions are handled so as to minimize foaming and are kept relatively concentrated
air-water; glass; plastic
long-term storage: processes such as … and … must be prevented when dealing with purified protein samples. protein solutions are sometimes stored under nitrogen/argon gas and/or are frozen at -80 or -196 degrees C.
slow oxidation; microbial contamination
the rate of product formation is proportional to the … present
amount of enzyme
proteins are quantified by …
… reaction: the product of the enzymatic reaction may be converted, by the action of another enzyme, to an easily quantified substance.
assays; coupled enzymatic reaction
… use antibodies which bind specifically to the original protein antigen
immunoassays
…: the protein is indirectly detected by determining the degree to which it competes with a radioactively labeled standard for binding to the antibody. another technique, the … assay, has many variations
radioimmunoassay; enzyme-linked immunosorbent assay
the concentration of a substance in solution can be measured by … a solution containing a solute that absorbs light does so according tot he Beer-Lambert law, A = log (I0/I) = Ecl, where A is the solute’s absorbance (its …), I0 is the intensity of the incident light at a given wavelength, I is its transmitted intensity, e is the … (alternatively, the …) of the solute, c is its concentration, and l is the length of the light path in centimeters
absorbance spectroscopy; optical density; absorptivity; extinction coefficient
if a protein has a … that absorbs in the visible region of the spectrum, this absorbance can be used to assay for the presence of the protein in a mixture of other proteins
chromophore
proteins are purified by … procedures. in a series of independent steps, the various physicochemical properties of the protein of interest are used to separate it progressively from other substances. the idea is not to minimize the loss of the desired proteins, but to eliminate … the other components of the mixture so that only the required substance remains
fractionation; selectively
the solubility of a protein at low ion concentrations increases as salt is added ,a phenomenon called … the additional ions shield the proteins’ multiple ionic charges, thereby weakening the attractive forces between individual protein molecules
salting in
as more salt is added, the solubility of the protein again decreases: … effect, primarily a result of the competition between the added salt ions and the other dissolved solutes for molecules of solvent
salting out
since different proteins have different ionic and hydrophobic compositions and thereby precipitate at different salt concentrations, … is the bases of one of the most commonly used protein purification procedures
salting out
the pH may be adjusted to approximate the isoelectric point of the desired protein because a protein is least soluble when its ..
net charge is zero
)in most … procedures, a mixture of substances to be fractionated is dissolved in a liquid (the mobile phase) and percolated through a colum containing a porous solid matrix (the stationary phase). as solutes flow through the column, they interact with the stationary phase and are retarded.
chromatographic
.… employs automated systems with precisely applied samples, controlled flow rates at high pressures, a chromatographic matrix of glass/plastic beads, and online sample detection
high performance liquid chromatography (HPLC)
anions bind to cationic groups on …, and cations bind to anionic groups on … in ion exchange chromatogrphy
anion exchangers; cation exchangers
Perhaps the most frequently used anion exchanger is a matrix with attached …` groups, and the most frequently used cation exchanger is a matrix bearing … groups.
diethylaminoethyl (DEAE); carboxymethyl (CM)
proteins and other … (polyionic polymers) that bear both positive and negative charges can bind to both cation and anion exchangers
polyelectrolytes
the binding affinity of a particular protein depends on the presence of other ions that compete with the protein for … and on the … of the solution, which influences the net charge of the protein
binding to the ion exchanger; pH
for ion exchange: as the column is washed with buffer, proteins with relatively low affinities for the ion exchanger move through the column … than those that bind with higher affinities. the column effluent is collected in a series of fractions. proteins that bind tightly to the ion exchanger can be … (washed through the column) by applying a buffer, called the …, that has a higher salt concentration/pH that reduces the affinity with which the matrix binds the protein
faster; eluted; eluant
in most cases, for ion exchange, the separation is enhanced by gradually increasing the eluant’s … or .. change over the course of the elution
salt concentration; pH
in … the matrix material is lightly substituted with octyl/phenyl groups. at high salt concentrations, nonpolar groups on the surface of proteins interact with the hydrophobic groups; that is, both types of groups are … by the polar solvent (hydrophobic effects are augmented by increased ionic strength). eluant typically aqueous buffer with decreasing salt []s, increasing concentrations of detergent, or changes in pH
hydrophobic interaction chromatography; excluded
in …. (also called … or …), molecules are separated according to their size and shape. stationary phase consists of gel beads containing pores that span a relatively narrow size range. the molecules that are too lareg to pass through the pores are excluded from the solvent volume inside the gel beads large molecules traverse the column more … than small molecules that pass through the pores
gel filtration chromatography; size exclusion; molecular sieve chromatography; rapidly
a striking characteristic of many proteins is their ability to bind specific molecules tightly but noncovalently. this property can be used to purify such proteins by … in this technique, a molecule (ligand) that specifically binds to the protein of interest is covalently attached to an inert matrix
affinity chromatography;
when an impure protein’s solution is passed through chromatographic material in affinity chromatography, the desired protein binds to the …, whereas other substances are washed through the column with the buffer. the great advantage of affinity chromatography is its ability to exploit the desired protein’s unique … properties rather than the small differences in physicochemical properties between proteins exploited by other chromatographic methods
immobilized ligand; biochemical
in …, an antibody is attached to the matrix to purify the protein against which the antibody was raised. in all cases, the ligand must have an affinity high enough to capture the protein of interest, but not so high as to prevent the proteins’ subsequent release without denaturing it
immunoaffinity chromatography
in …, a divalent metal ion such as Zn2+ or Ni2+ is attached to the chromatographic matrix so that proteins bearing metal chelating groups (e.g. multiple HIs side chains) can be retained
metal chelate affinity chromatography
metal chelate chroma:
recombinant DNA techniques can be used to append a segment of six consecutive His residues, known as a …, to the N- or C- terminus of the polypeptide to be isolated, creating a metal ion-binding site allowing the recombinant protein to be purified
His tag
… employs migration of ion sin an electrif field. typically carried out in polyacrylamide gels with a characteristic pore size, so the molecular separations are based on …. (size and shape) as well as … (electric charge)
polyacrylamide gel electrophoresis (PAGE); gel filtration; electrophoretic mobility
if the proteins in a sample are radioactive, the gel can be dried and clamped over a sheet of X-ray film. after a time, the film is developed and the resulting .. .shows the positions of the radioactive components by blackening of the film
autoradiograph
if an antibody to a protein of interest is available, it can be used to specifically detect the protein on a gel in the presence of many other proteins, a process called … or …
immunoblotting; Western blotting
SDS (sodium dodecyl sulfate) treated proteins have similar shapes and charge-to-mass ratios. SDS PAGE therefore separates proteins purely by …
the possibility that subunits are linked by disulfide bonds can be tested by preparing samples for SDS PAGE in the presence and absence of a reducing agent, such as …, that breaks those bonds
gel filtration effects; 2-mercaptoethanol
… overcomes the disadvantages of gel electrophoresis, which can take up to several hrs and is difficult to quantitate and automate
capillary electrophoresis
capillary electrophoresis is a technique in which electrophoresis is carried out in very thin .., which rapidly dissipate heat and therefore permit the use of very high .., which reduces separation times to a few mins
capillary tubes; electric fields
if a mixture of protein is electrophoresed through a solution/gel that has a stable pH gradient in which the pH increases smoothly from anode to cathode, each protein will migrate to the position in the pH gradient corresponding to its … this type of electrophoresis is called … (IEF)
pI; isolectric focusing
iEF can be combined with SDS-PAGE in an extremely powerful separation technique named … First, sample is subjected to IEF in one direction, and then subjected to SDS PAGE in the perpendicular direction
two-dimensional (2D) gel electrophoresis
…: field of study that involves cataloging all of a cell’s expressed proteins with emphasis on their quantitation, localization, modifications, interactions, and activities
proteomics
only when subjected to enormous … do macromolecules begin to sediment, much like grains of sand in water.–> achieved by …
accelerations; ultracentrifuge
the rate at which a particle sediments in the ultracentrifuge is related to its …
mass
a protein’s sedimentation coefficient is usually expressed in units of 10^-13 s, which are known as …
Svedbergs
typically, sedimentation is carried out in a solution of an inert substance in which the concentration, and therefore the density, of the solution increases from the top to the bottom of the centrifuge–> …
density gradient
knowledge of a protein’s amino acid sequence is a prereq for determining its … and is essential for understanding its molecular ….
three dimensional structure; mechanism of action
sequence comparisons among analogous proteins from different species yield insights into … and reveal .. among the proteins and the organisms that produce them
protein function; evolutionary relationships
many inherited diseases are caused by mutations that result in an … in a protein. amino acid sequence analysis can assist in the development of diagnostic tests and effective therapies
amino acid change
the protein must be broken down into fragments small enough to be …, and the primary structure of the intact proteins is then reconstructed from the sequences of …
individually sequenced; overlapping fragments
… reveals the number of different types of subunits–> each polypeptide chain has an N-terminal residue. identifying this end group can establish the number of … polypeptides in a protein
N-terminal analysis; chemically distinct
disulfide bonds between cys residues must be cleaved to …-if they are disulfide-linked-and to ensure that polypeptide chains are fully …
separate polypeptide chains; linear
disulfide bonds can be reductively cleaved by treating them with 2-mercaptoethanol or another … the resulting free sulfhydrl groups are then alkylated, usually by treatment with …, to prevent the re-formation of disulfide bonds through oxidation by …
mercaptan; iodoacetate; O2
polypeptides that are longer than … to … residues cannot be directly sequenced and must therefore be cleaved, either enzymatically or chemically, to specific fragments that are small enough to be sequenced. various … (enzymes that catalyze the hydrolysis of internal peptide bonds, as opposed to …, which catalyze the hydrolysis of N- or C- terminal residues) can be used to fragment polypeptides
25; 100; endopeptidases; exopeptidases
collectively, endo and exopeptidases are called …
proteases
… has the greatest specificity and is therefore the most valuable member of the arsenal of endopeptidases used to fragment polypeptides. it cleaves peptide bonds on the C side of Arg and Lys if the next residue is not …
trypsin; Pro
through … (by adjusting rxn conditions and limiting rxn times), less specific endopeptidases can yield a set of discrete, nonoverlapping fragments.
several chemical reagents promote peptide bond cleavage at specific residues. e.g. …
limited proteolysis; cyanogen bromide (CNBr)
once the peptide fragments formed through specific cleavage rxns have been isolated, their amino acid sequences can be determined through repeated cycles of …
edman degradation
the two steps of the peptide cleavage cleavage process take place under different conditions. hence, the amino acid sequence of a polypeptide chain can be determined from the … inward by subjecting the polypeptide to repeated cycles of Edman degradation and, after every cycle, identifying the newly liberated …
N-terminus; PTH-amino acid
up to … residues can be identified through edman degradation before the cumulative effects of incomplete rxns, side rxns, and peptide loss make further amino acid identification unreliable
100
… has emerged as the dominant technique for characterizing and sequencing proteins. accurately measures the mass/charge ratio for ions in the gas phase
mass spectrometry
…: solution of a macromolecule is sprayed from a narrow capillary tube maintained at high voltage forming fine, highly charged droplets from which the solvent rapidly evaporates
electro-spray ionization (ESI)
using ESI, ions are directed into the mass spec, which measures their m/z values. consequently, determining an ion’s z permits it molecular mass to be determined with far greater … than by any other method
accuracy
…: unidentified protein is cleaved into define fragments and their masses are determined. members of a database of proteins of known sequences are theoretically subjected to the same cleavage method and the masses of the resulting peptides calculated. a match between the experimental and a theoretical set of masses identifies the protein
protein mass fingerprinting
short polypeptides can be directly sequenced through the use of a …
tandem mass spectrometer (two mass specs coupled in series)
by comparing the molecular masses of successively larger members of a family of fragments, the … and therefore the …of successive amino acid residues in a polypeptide are determined
molecular masses; identities
protein sequence information is no less valuable when the base sequence of the corresponding gene is known, because a protein sequence provides info about … that is not revealed by nucleic acid sequencing
protein structure
… is a component of the mitochondrial electron transport system that is closely related among eukaryotes
cytochrome c
evolutionary theory indicates that related species have evolved from a common ancestor, so it follows that the genes specifying each of their proteins must likewise have evolved from the … in that ancestor
corresponding gene
In general, comparisons of the primary structures of … proteins (evolutionarily related proteins) indicate which of the protein’s residues are essential to its function, which are less significant, and which have little specifi c function
homologous
… residue: uniquely suited to some essential unction of the protein
invariant
… substituted: residue that can be replaced with others of similar characteristics
conservatively
…: a particular amino acid position may tolerate many diff residues, indicating that the functional requirements of that position are nonspecific
hypervariable
…: random nature of mutational processes will, in time, change such a protein in ways that do not significantly affect its function
neutral drift
analysis of a large number of proteins indicates that evolutionary conserved sequences are often segments of about … to … residues, called …
40; 200; domains
domains whose sequences are more than …% identical usually have the same function; domains with less than about …% sequence identity usually perform different roles
40; 25
homologous proteins with the same function in different species are said to be …
orthologous
within a species, similar proteins arise through …, an aberrant genetic recombination event in which one member of a chromosome pair acquires both copies of the primordial gene
gene duplication
gene duplication is a particularly efficient mode of evolution bc one copy of the gene evolves a … through natural selection while its counterpart continues to direct the synthesis of the original protein–> two independently evolving genes derived from a duplication event are said to be
new function; paralogous
… family of proteins (hemoglobin, myoglobin, etc) is an example of evolution through gene duplication and divergence
globin
human genome contains relics of genes that aren’t expressed –> .. that can be considered the dead ends of protein evolution
pseudogenes
rates of evolution vary among proteins. this does not imply that the rates of mutation of the DNAs specifying those proteins differ, but rather that the rate at which mutations are … varies
accepted into a protein
…: a protein that binds to DNA in eukaryotes –> highly conserved protein
histone H4
the … are about 20-residue fragments that are cleaved from the vertebrate protein … to induce blood clotting. once they have been removed, they are discarded, so that they are subject to little selective pressure to maintain their amino acids equences
fibrinopeptides; fibrinogen
the rate of protein evolution also depends on the protein’s …
structural stability
…: gene segments encoding protein domains are copied and inserted into other positions in a genome to generate new genes that encode proteins with novel sequences
domain shuffling
functions of individual domains not always known: some appear to have discrete activities, such as catalyzing certain rxns/binding particular molecules, but others may just be … or … for other domains
spacers; scaffolding
