• Proteins (working molecules of a cell) carry out a program of activities encoded by genes

• Gradually many of the primitive proteins evolved into a wide array of enzymes cable of catalyzing an incredible range of intracellular and extracellular chemical reactions

• The functional design of proteins have “moving” parts and are capable of transmitting various forces and energy

3.1 - Hierarchical Structure of Proteins

• There are 20 different amino acids construed by polymerization into linear chains

• A protein is only able to function correctly when it is in its three-dimensional structure or conformation

The Primary Structure of a Protein Is Its Linear Arrangement of Amino Acids

• The primary structure of a protein is simply the linear arrangement or sequence of amino acids that residues compose

• Short-chain of amino acids linked by peptide bonds and having a defined sequence is called peptide

  • Fewer than 20-30 amino acids

• Long-chains are referred to as polypeptides

  • Can contain as many as 4000

• The term protein is usually reserved for polypeptides

• Proteins and peptides have been considered natural products of a cell

• The size of a protein (polypeptide) is represented by daltons

• Predicted proteins encoded by the yeast genome have an average molecular weight of 52.728 and contain an average of 466 amino acids

• The average molecular weight of amino acids in proteins is 113, taking into account their average relative abundance

Secondary Structure Are the Core Elements of Protein Architecture

• The second level in the hierarchy of protein structure consists of the various spatial arrangements resulting from the folding of localized parts of a polypeptide chain

  • These arrangements are referred to as secondary structures

• A single polypeptide may exhibit multiple types of secondary structure depending on its sequence

• The helix in a polypeptide segment folded into a helix

  • The carbonyl oxygen atom of each peptide bond is hydrogen-bonded to the amide hydrogen atom of the amino acid four residues toward the C-terminus

• This periodic arrangement of bonds confer a directionality on the helix because all the hydrogen-bond donors have the same orientation

• The hydrophobic or hydrophilic quality of the helix is determined entirely by the side chains

  • The polar groups of the peptide backbone are already engaged in hydrogen bonding in the helix

• The B Sheet is another type of secondary structure, the B sheet, consists of laterally packed B strands

• Each B strand is a short (5- to 8-residue), nearly fully extended polypeptide segment

• This structure is also called a B pleated sheet in a pleated sheet

  • Adjacent B strands can be oriented in the same (parallel) or opposite (antiparallel) directions with respect to each other

• Turns are composed of three or four residues, turns are located on the surface of a protein, forming sharp bends that redirect the polypeptide backbone back toward the interior

Overall Folding of a Polypeptide Chain Yields Its Tertiary Structure

• Tertiary Structure refers to the overall conformation of a polypeptide chain—that is, the three-dimensional arrangement of all its amino acid residues

• In contrast with secondary structures, which are stabilized by hydrogen bonds

  • Tertiary structure is primarily stabilized by hydrophobic interactions between the nonpolar side chains, hydrogen bonds between polar side chains, and peptide bonds

• These stabilizing forces hold elements of secondary structure— helices, strands, turns, and random coils—compactly together

• This variation in structure has important consequences on the function and

• regulation of proteins

• The simplest way to represent a three-dimensional structure is to trace the course of the backbone atoms with a solid line

• Another type of representation uses common shorthand symbols for depicting a secondary structure

• None of these three ways of representing protein structure convey much information about the protein surface

Motifs Are Regular Combinations of Secondary Structures

• Particular combinations of secondary structures, called motifs or folds, build up the tertiary structure of a protein

• Motif, also called the EF-hand, has been found in more than 100 calcium-binding proteins

• Many proteins, especially fibrous proteins, self-associate into oligomers by using a third motif, the coiled-coil

  • In these proteins, each polypeptide chain contains helical segments in which the hydrophobic residues, although apparently randomly arranged, are in a regular pattern (a repeated heptad sequence)

• The overall helical structure is amphipathic

Structural and Functional Domains Are Modules of Tertiary Structure

• The tertiary structure of proteins larger than 15,000 MW is typically subdivided into distinct regions called domains

• Large proteins, domains can be recognized in structures determined by x-ray crystallography or in images captured by electron microscopy

• A structural domain consists of 100–150 residues in various combinations of motifs

• Domains are sometimes defined in functional terms on the basis of observations that an activity of a protein is localized to a small region along its length

• DNA encoding a protein can be subjected to mutagenesis so that segments of the protein’s backbone are removed or changed

• The organization of large proteins into multiple domains illustrates the principle that complex molecules are built from simpler components

• The epidermal growth factor (EGF) domain is one example of a module that is present in several proteins

• EGF is a small, soluble peptide hormone that binds to cells in the embryo and in the skin and connective tissue in adults, causing them to divide

• EGF modules are also present in other proteins and are liberated by proteolysis

• Besides the EGF domain, these proteins contain domains found in other proteins

Proteins Associate into Multimeric Structures and Macromolecular Assemblies

• Multimeric proteins consist of two or more polypeptides or subunits

• The fourth level of structural organization, quaternary structure, describes the number (stoichiometry) and relative positions of the subunits in multimeric proteins

• Other multimeric proteins can be composed of any number of identical or different subunits

• Enzymes in the same pathway may be associated as subunits of a large multimeric protein within the cell, thereby increasing the efficiency of pathway operation

• The highest level of protein structure is the association of proteins into macromolecular assemblies

  • Such structures are very large, exceeding 1 mDa in mass, approaching 30–300nm in size, and containing tens to hundreds of polypeptide chains, as well as nucleic acids in some cases

• Macromolecular assemblies with a structural function

  • Include the capsid that encases the viral genome and bundles of cytoskeletal filaments that support and give shape to the plasma membrane

• The transcription factors and promoter-binding proteins correctly position a polymerase molecule at a promoter

  • The DNA site determines where transcription of a specific gene begins

Members of Protein Families Have a Common Evolutionary Ancestor

• Myoglobin and hemoglobin (the oxygen-carrying proteins in muscle and blood) respectively

  • Provided early evidence that function derives from three-dimensional structure, which in turn is specified by the amino acid sequence

• Subsequent sequencing of myoglobin and the hemoglobin subunits revealed that many identical or chemically similar residues are found in identical positions throughout the primary structures of both proteins

• Proteins that have a common ancestor are referred to as homologs

• The main evidence for homology among proteins

3.2 - Folding, Modification, and Degradation of Proteins

• A polypeptide chain is synthesized by a complex process called translation

• Incorrectly folded proteins usually lack biological activity and, in some cases, may actually be associated with disease

• Protein misfolding is suppressed by two distinct mechanisms

  • Cells have systems that reduce the chances for misfolded proteins to form

  • Any misfolded proteins that do form, as well as cytosolic proteins no longer needed by a cell, are degraded by a specialized cellular garbage disposal system

The Information for Protein Folding Is Encoded in the Sequence

• Any polypeptide chain containing n residues could, in principle, fold into 8n conformations

• Thermal energy from heat, extremes of pH that alter the charges on amino acid side chains

  • Chemicals such as urea or guanidine hydrochloride at concentrations of 6–8 M can disrupt the weak noncovalent interactions that stabilize the native conformation of a protein

• Sufficient information must be contained in the protein’s primary sequence to direct correct refolding

Folding of Proteins in Vivo Is Promoted by Chaperones

• Protein folding occurs in vitro, only a minority of unfolded molecules undergo complete folding into the native confirmation within a few minutes

• More than 95 percent of the proteins present within cells have been shown to be in their native conformation

  • Despite high protein concentrations (200–300 mg/ml), which favor the precipitation of proteins in vitro

• Molecular chaperones consist of Hsp70 and its homologs: Hsp70 in the cytosol and mitochondrial matrix, BiP in the endoplasmic reticulum, and DnaK in bacteria

• Molecular chaperones are thought to bind all nascent polypeptide chains as they are being synthesized on ribosomes

• In bacteria, 85 percent of the proteins are released from their chaperones and proceed to fold normally

  • An even higher percentage of proteins in eukaryotes follow this pathway

Many Proteins Undergo Chemical Modification of Amino Acid Residues

• Nearly every protein in a cell is chemically modified after its synthesis on a ribosome

• An important modification is the phosphorylation of serine, threonine, tyrosine, and histidine residues

• The side chains of asparagine, serine, and threonine are sites for glycosylation, the attachment of linear and branched carbohydrate chains

• Other post-translational modifications found in selected proteins include the hydroxylation of proline and lysine residues in collagen, the methylation of histidine residues in membrane receptors, and the -carboxylation of glutamate in prothrombin, an essential blood-clotting factor

Peptide Segments of Some Proteins are Removed After Synthesis

• After their synthesis, some proteins undergo irreversible changes that do not entail changes in individual amino acid residues

• This type of post-translational alteration is sometimes called processing

• An unusual and rare type of processing, termed protein self-splicing, takes place in bacteria and some eukaryotes

• This process is analogous to editing film: an internal segment of a polypeptide is removed and the ends of the polypeptide are rejoined

• Proteolytic processing, protein self-splicing is an autocatalytic process, which proceeds by itself without the participation of enzymes

Ubiquitin Marks Cytosolic Proteins for Degradation in Proteasomes

• The activity of a cellular protein depends on the amount present, which reflects the balance between its rate of synthesis and the rate of degradation in the cell

• The life span of intracellular proteins varies from as short as a few minutes for mitotic cyclins

  • Which help regulate passage through mitosis, to as long as the age of an organism for proteins in the lens of the eye

• Eukaryotic cells have several intracellular proteolytic pathways for degrading misfolded or denatured proteins

  • Normal proteins whose concentration must be decreased and extracellular proteins are taken up by the cell

• The major intracellular pathway is degradation by enzymes within lysosomes, membrane-limited organelles whose acidic interior is filled with hydrolytic enzymes

• The immune system also makes use of the ubiquitin-mediated pathway in the response to altered self-cells, particularly virus-infected cells

• Viral proteins within the cytosol of infected cells are ubiquitinated and then degraded in proteasomes specially designed for this role

Digestive Proteases Degrade Dietary Proteins

• The major extracellular pathway for protein degradation is the system of digestive proteases that breaks down ingested proteins into peptides and amino acids in the intestinal tract

• Three classes of proteases function indigestion

  • Endeoroteases

  • Exopeptidases

  • Peptidases

• To protect a cell from degrading itself, endoproteases and carboxypeptidases are synthesized and secreted as inactive forms (zymogens): pepsin by chief cells in the lining of the stomach; the others by pancreatic cells

Alternatively Folded Proteins Are Implicated in Slowly Developing Diseases

• Recent evidence suggests, that a protein may fold into an alternative three-dimensional structure as the result of mutations

  • Inappropriate post-translational modification, or other as-yet-unidentified reasons

• Such “misfolding” not only leads to a loss of the normal function of the protein but also marks it for proteolytic degradation

3.3 - Enzymes and the Chemical Work of Cells

• The function of nearly all proteins depends on their ability to bind other molecules, or ligands, with a high degree of specificity

Specificity and Addinity of Protein-Ligand Binding Depend on Molecular Complementarity

• Specificity refers to the ability of a protein to bind one molecule in preference to other molecules

• Affinity refers to the strength of binding

• The stronger the interaction between a protein and ligand, the lower the value of Kd

• For high-affinity and highly specific interactions to take place the shape and chemical surface of the binding site must be complementary to the ligand molecule

  • A property termed molecular complementarity

• The ability of proteins to distinguish different molecules is perhaps most highly developed in the blood proteins called antibodies

  • Which animals produce in response to antigens, such as infectious agents, and certain foreign substances

• The presence of an antigen causes an organism to make a large number of different antibody proteins

  • Each of which may bind to a slightly different region or epitope of the antigen

• Antibodies act as specific sensors for antigens, forming antibody-antigen complexes that initiate a cascade of protective reactions in cells of the immune system

• The specificity of antibodies is so precise that they can distinguish between the cells of individual members of a species and in some cases can distinguish between proteins that differ by only a single amino acid

Enzymes Are Highly Efficient and Specific Catalysts

• In contrast with antibodies, which bind and simply present their ligands to other components of the immune system, enzymes promote the chemical alteration of their ligands, called substrates

• Two striking properties of enzymes enable them to function as catalysts under the mild conditions present in cells: their enormous catalytic power and their high degree of specificity

• Approximately 3700 different types of enzymes, each of which catalyzes a single chemical reaction or set of closely related reactions, have been classified in the enzyme database

• Certain enzymes are found in the majority of cells because they catalyze the synthesis of common cellular products

• Other enzymes are present only in a particular type of cell because they catalyze chemical reactions unique to that cell type

• Most enzymes are located within cells, some are secreted and function in extracellular sites such as the blood, the lumen of the digestive tract, or even outside the organism

• The catalytic activity of some enzymes is critical to cellular processes other than the synthesis or degradation of molecules

An Enzyme’s Active Site Binds Substrates and Carries Out Catalysis

• Certain amino acid side chains of an enzyme are important in determining its specificity and catalytic power

• In the native conformation of an enzyme, these side chains are brought into proximity, forming the active site

• Active sites thus consist of two functionally important regions

  • One that recognizes and binds the substrate (or substrates)

  • One that catalyzes the reaction after the substrate has been bound

• For some enzymes, the catalytic region is part of the substrate-binding region; in others, the two regions are structurally as well as functionally distinct

• The active site of protein kinase A is located in the 240-residue “kinase core” of the catalytic subunit

• Substrate Binding by Protein Kinases; the structure of the ATP-binding site in the catalytic kinase core complements the structure of the nucleotide substrate

• The adenine ring of ATP sits snugly at the base of the cleft between the large and the small domains

• The catalytic core of protein kinase A exists in an “open” and “closed” conformation

• When the active site is occupied by the substrate, the domains move together into the closed position

• Phosphoryl Transfer by Protein Kinases; after substrates have bound and the catalytic core of protein kinase A has assumed the closed conformation, the phosphorylation of a serine or threonine residue on the target peptide can take place

Vmax and Km Characterize and Enzymatic Reaction

• The catalytic action of an enzyme on a given substrate can be described by two parameters: Vmax, the maximal velocity of the reaction at saturating substrate concentrations, and Km (the Michaelis constant), a measure of the affinity of an enzyme for its substrate

• The concentrations of the various small molecules in a cell vary widely, as do the Km values for the different enzymes that act on them

Enzymes in a Common Pathway Are Often Physically Associated with One Another

• Enzymes taking part in a common metabolic process are generally located in the same cellular compartment

• In the simplest such mechanism, polypeptides with different catalytic activities cluster closely together as subunits of a multimeric enzyme or assemble on a common “scaffold”

• In some cases, separate proteins have been fused together at the genetic level to create a single multi-domain, multi-functional enzyme

3.4 - Molecular Motors and the Mechanical Work of Cells

• A common property of all cells is motility, the ability to move in a specified direction

• Many cell processes exhibit some type of movement at either the molecular or the cellular level; all movements result from the application of a force

• Differently, materials within a cell are transported in specific directions and for longer distances

Molecular Motors Convert Energy into Motion

• At the nanoscale of cells and molecules, movement is affected by many different forces from those in the macroscopic world

• To generate the forces necessary for many cellular movements, cells depend on specialized enzymes commonly called motor proteins

• Motor proteins generate either linear or rotary motion

• This latter group comprises the myosins, kinesins, and dyneins, linear motor proteins that carry attached “cargo” with them as they proceed along either microfilaments or microtubules

• DNA and RNA polymerases also are linear motor proteins because they translocate along with DNA during replication and transcription

• The torque generated by the stator rotates an inner ring of proteins and the attached flagellum

• Interactions between the subunit and the B subunits directs the synthesis of ATP

• The motor proteins that attach to cytoskeletal fibers also bind to and carry along with cargo as they translocate

• The cargo in muscle cells and eukaryotic flagella consist of thick filaments and B tubules

All Myosins Have Head, Neck, and Tail Domains with Distinct Functions

• Consider myosin II, which moves along actin filaments in muscle cells during contraction

• Other types of myosin can transport vesicles along actin filaments in the cytoskeleton. Myosin II and other members of the myosin superfamily are composed of one or two heavy chains and several light chains

• The heavy chains are organized into three structurally and functionally different types of domains

• Results of studies of myosin fragments produced by proteolysis helped elucidate the functions of the domains

Conformational Changes in the Myosin Head Could APT Hydrolysis to Movement

• The results of studies of muscle contraction provided the first evidence that myosin heads slide or walk along actin filaments

• Unraveling the mechanism of muscle contraction was greatly aided by the development of in vitro motility assays and single-molecule force measurements.

• One assumption in this model is that the hydrolysis of a single ATP molecule is coupled to each step taken by a myosin molecule along an actin filament

• Myosin undergoes a series of events during each step of movement

• In the course of one cycle, myosin must exist in at least three conformational states: an ATP state unbound to actin

  • An ADP-Pi state bound to actin, and a state after the power-generating stroke has been completed

• Results of structural studies of myosin in the presence of nucleotides and nucleotide analogs that mimic the various steps in the cycle indicate that the binding and hydrolysis of a nucleotide cause a small conformational

  • Change in the head domain that is amplified into a large movement of the neck region

• Homologous switch, converter, and lever arm structures in kinesin are responsible for the movement of kinesin motor proteins along microtubules

• The structural basis for dynein movement is unknown because the three-dimensional structure of dynein has not been determined

3.5 - Common Mechanisms for Regulating Protein Function

• The catalytic activity of enzymes or the assembly of a macromolecular complex is so regulated that the amount of reaction product or the appearance of the complex is just sufficient to meet the needs of the cell

• Resulting, the steady-state concentrations of substrates and products will vary, depending on cellular conditions

• One of the most important mechanisms for regulating protein function entails allostery

• Allostery refers to any change in a protein’s tertiary or quaternary structure or both induced by the binding of a ligand

  • Which may be an activator, inhibitor, substrate, or all three

Cooperative Binding Increases a Protein’s Response to Small Changes in Land Concentration

• When a protein binds several molecules of one ligand, the binding is graded; that is, the binding of one ligand molecule affects the binding of subsequent ligand molecules

• This type of allostery often called cooperativity

  • Permits many multisubunit proteins to respond more efficiently to small changes in ligand concentration than would otherwise be possible

• In positive cooperativity, sequential binding is enhanced; in negative cooperativity, sequential binding is inhibited

Ligand Binding Can Induce Allosteric Release of Catalytic Subunits or Transition to a State with Different Activity

• Each regulatory subunit contains a pseudosubstrate sequence that binds to the active site in a catalytic subunit

• Inactive protein kinase A is turned on by cyclic AMP (cAMP), a small second-messenger molecule

• When the signaling ceases and the cAMP level decreases, the activity of protein kinase A is turned off by reassembly of the inactive tetramer

• The binding of cAMP to the regulatory subunits exhibits positive cooperativity

  • Thus small changes in the concentration of this allosteric molecule produce a large change in the activity of protein kinase A

• Many multimeric enzymes undergo allosteric transitions that alter the relation of the subunits to one another but don’t cause dissociation as in protein kinase A

Calcium and GTP Are Widely Used to Modulate Protein Activity

• Calmodulin-Mediated Switching: The concentration of Ca2 free in the cytosol is kept very low (≈107 M) by membrane transport proteins that continually pump Ca2 out of the cell or into the endoplasmic reticulum

• The rise in cytosolic Ca2 is sensed by Ca2-binding proteins, particularly those of the EF-hand family, all of which contain the helix-loop-helix motif

• The prototype EF-hand protein, calmodulin is found in all eukaryotic cells and may exist as an individual monomeric protein or as a subunit of a multimeric protein

• Calmodulin and similar EF-hand proteins thus function as switch proteins, acting in concert with Ca2 to modulate the activity of other proteins

• Switching Mediated by Guanine Nucleotide–Binding: Proteins Another group of intracellular switch proteins constitutes the GTPase superfamily

• These proteins include monomeric Ras protein and the G subunit of the trimeric G proteins

  • Both Ras and G are bound to the plasma membrane, function in cell signaling, and play a key role in cell proliferation and differentiation

• All the GTPase switch proteins exist in two forms: (1) an active (“on”) form with bound GTP (guanosine triphosphate) that modulates the activity of specific target proteins and (2) an inactive (“off”) form with bound GDP (guanosine diphosphate)

• The subsequent exchange of GDP with GTP to regenerate the active form occurs even more slowly

• Activation is temporary and is enhanced or depressed by other proteins acting as allosteric regulators of the switch protein

Cyclic Protein Phosphorylation and Dephoshoroylation Regulate Many Cellular Functions

• One of the most common mechanisms for regulating protein activity is phosphorylation, the addition, and removal of phosphate groups from serine, threonine, or tyrosine residues

• Protein kinases catalyze phosphorylation, and phosphatases catalyze dephosphorylation

• Phosphorylation changes a protein’s charge and generally leads to a conformational change; these effects can significantly alter ligand binding by a protein, leading to an increase or decrease in its activity

• All classes of proteins (including structural proteins, enzymes, membrane channels, and signaling molecules) are regulated by kinase/phosphatase switches

• Different protein kinases and phosphatases are specific for different target proteins and can thus regulate a variety of cellular pathways

• Some of these enzymes act on one or a few target proteins, whereas others have multiple targets

Proteolytic Cleavage Irreversibly Activates or Inactivates Some Proteins

• The regulation of some proteins is by a distinctly different mechanism: the irreversible activation or inactivation of protein function by proteolytic cleavage

• This mechanism is most common in regard to some hormones (e.g., insulin) and digestive proteases

• Enterokinase, an aminopeptidase secreted from cells lining the small intestine, converts trypsinogen into trypsin, which in turn cleaves chymotrypsinogen to form chymotrypsin

Higher-Order Regulation Includes Control of Protein Location and Concentration

• The activities of proteins are extensively regulated in order that the numerous proteins in a cell can work together harmoniously

• The normal functioning of a cell requires the segregation of proteins to particular compartments such as the mitochondria, nucleus, and lysosomes

• In addition to compartmentation, cellular processes are regulated by protein synthesis and degradation

• When the cell faces increased demand (e.g., the appearance of the substrate in the case of enzymes, stimulation of B lymphocytes by antigen), the cell responds by synthesizing new protein molecules

Purifying, Detecting, and Characterizing Proteins

• Protein must be purified before its structure and the mechanism of its action can be studied

  • Because proteins vary in size, charge, and water solubility, no single method can be used to isolate all proteins

• Any molecule, whether protein, carbohydrate, or nucleic acid, can be separated, or resolved, from other molecules on the basis of their differences in one or more physical or chemical characteristics

• The larger and more numerous the differences between two proteins, the easier and more efficient their separation

Centrifugation Can Separate Particles and Molecules That Differ in Mass or Density

• The first step in a typical protein purification scheme is centrifugation

• The principle behind centrifugation is that two particles in suspension (cells, organelles, or molecules) with different masses or densities will settle to the bottom of a tube at different rates

• Proteins vary greatly in mass but not in density

• Unless a protein has an attached lipid or carbohydrate, its density will not vary by more than 15 percent from 1.37 g/cm3, the average protein density

• Heavier or more dense molecules settle, or sediment, more quickly than lighter or less dense molecules

• A centrifuge speeds sedimentation by subjecting particles in suspension to centrifugal forces as great as 1,000,000 times the force of gravity g

  • Which can sediment particles as small as 10 kDa

• Centrifugation is used for two basic purposes: (1) as a preparative technique to separate one type of material from others and (2) as an analytical technique to measure physical properties (e.g., molecular weight, density, shape, and equilibrium binding constants) of macromolecules

• Differential Centrifugation: The most common initial step in protein purification is the separation of soluble proteins from insoluble cellular material by differential centrifugation

• Rate-Zonal Centrifugation: On the basis of differences in their masses, proteins can be separated by centrifugation through a solution of increasing density called a density gradient

• Although the sedimentation rate is strongly influenced by particle mass

  • Rate-zonal centrifugation is seldom effective in determining precise molecular weights because variations in shape also affect sedimentation rate

Electrophoresis Separates Molecules on the Basis of Their Charge: Mass Ratio

• Electrophoresis is a technique for separating molecules in a mixture under the influence of an applied electric field

• Dissolved molecules in an electric field move, or migrate, at a speed determined by their charge: mass ratio

• SDS-Polyacrylamide Gel Electrophoresis: Because many proteins or nucleic acids that differ in size and shape have nearly identical charges: mass ratios

  • Electrophoresis of these macromolecules in solution results in little or no separation of molecules of different lengths

• But, successful separation of proteins and nucleic acids can be accomplished by electrophoresis in various gels (semisolid suspensions in water) rather than in a liquid solution

• Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels

• When a mixture of proteins is applied to a gel and an electric current is applied, smaller proteins migrate faster through the gel than do larger proteins

• Gels are cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously cross-linking the chains into a semisolid matrix

• In the most powerful technique for resolving protein mixtures, proteins are exposed to the ionic detergent SDS (sodium dodecyl sulfate) before and during gel electrophoresis

• SDS denatures proteins, causing multimeric proteins to dissociate into their subunits

  • All polypeptide chains are forced into extended conformations with similar charges: mass ratios

• Chains that differ in molecular weight by less than 10 percent can be separated by this technique

• Two-Dimensional Gel Electrophoresis: Electrophoresis of all cellular proteins through an SDS gel can separate proteins having relatively large differences in mass but cannot resolve proteins having similar masses (e.g., a 41-kDa protein from a 42-kDa protein)

• Commonly, this characteristic is the electric charge, which is determined by the number of acidic and basic residues in a protein

• In two-dimensional electrophoresis, proteins are separated sequentially, first by their charges and then by their masses

• A charged protein will migrate through the gradient until it reaches its isoelectric point (pI), the pH at which the net charge of the protein is zero

  • The technique called isoelectric focusing (IEF) can resolve proteins that differ by only one charge unit

• The sequential resolution of proteins by charge and mass can achieve excellent separation of cellular proteins

Liquid Chromatography Resolves Proteins by Mass, Charge, or Binding Affinity

• Another common technique for separating mixtures of proteins, as well as other molecules

  • Is based on the principle that molecules dissolved in a solution will interact (bind and dissociate) with a solid surface

• If the solution is allowed to flow across the surface, then molecules that interact frequently with the surface will spend more time-bound to the surface and move more slowly than molecules that interact infrequently with the surface

  • In this technique, called liquid chromatography

• Gel Filtration Chromatography: Proteins that differ in mass can be separated on a column composed of porous beads made from polyacrylamide, dextran (a bacterial polysaccharide), or agarose (a seaweed derivative)

• They spend some time within the large depressions that cover a bead’s surface

• Because smaller proteins can penetrate into these depressions more easily than can larger proteins

  • They travel through a gel filtration column more slowly than do larger proteins

• Ion-Exchange Chromatography: The second type of liquid chromatography, proteins are separated on the basis of differences in their charges

• This technique makes use of specially modified beads whose surfaces are covered by amino groups or carboxyl groups and carry either a positive charge (NH3) or a negative charge (COO) at neutral pH

• Proteins in a mixture carry various net charges at any given pH

• Affinity Chromatography: The ability of proteins to bind specifically to other molecules is the basis of affinity chromatography

  • In this technique, ligand molecules that bind to the protein of interest are covalently attached to the beads used to form the column

• An affinity column will retain only those proteins that bind the ligand attached to the beads; the remaining proteins, regardless of their charges or masses, will pass through the column without binding to it

Highly Specific Enzyme and Antibody Assays Can Detect Individual Proteins

• Purification of a protein, or any other molecule, requires a specific assay that can detect the molecule of interest in column fractions or gel bands

• Assay capitalizes on some highly distinctive characteristics of a protein: the ability to bind a particular ligand, to catalyze a particular reaction, or to be recognized by a specific antibody

• Chromogenic and Light-Emitting Enzyme Reactions: Many assays are tailored to detect some functional aspect of a protein

• Some enzyme assays utilize chromogenic substrates, which change colour in the course of the reaction

• Such chromogenic enzymes can also be fused or chemically linked to an antibody and used to “report” the presence or location of the antigen

• Western Blotting: A powerful method for detecting a particular protein in a complex mixture combines the superior resolving power of gel electrophoresis, the specificity of antibodies, and the sensitivity of enzyme assays

• Called Western blotting, or immunoblotting, this multistep procedure is commonly used to separate proteins and then identify a specific protein of interest

Radioisotopes Are Indispensable Tools for Detecting Biological Molecules

• A sensitive method for tracking a protein or other biological molecule is by detecting the radioactivity emitted from radioisotopes introduced into the molecule

• One atom in a radiolabeled molecule is present in a radioactive form, called a radioisotope

• Radioisotopes Useful in Biological Research: Hundreds of biological compounds (e.g., amino acids, nucleosides, and numerous metabolic intermediates) labeled with various radioisotopes are commercially available

• The specific activity of a labeled compound must be high enough that sufficient radioactivity is incorporated into cellular molecules to be accurately detected

• In most experiments, the former is preferable because they allow RNA or DNA to be adequately labeled after a shorter time of incorporation or require a smaller cell sample

• Labeled compounds (which a radioisotope replaces atoms normally present in the molecule) have the same chemical properties as the corresponding nonlabeled compounds

• Labeling Experiments and Detection of Radiolabeled Molecules: Whether labeled compounds are detected by autoradiography, a semiquantitative visual assay, or their radioactivity is measured in an appropriate “counter”

• In one use of autoradiography, a cell or cell constituent is labeled with a radioactive compound and then overlaid with a photographic emulsion sensitive to radiation

• Quantitative measurements of the amount of radioactivity in a labeled material are performed with several different instruments

• A combination of labeling and biochemical techniques and visual and quantitative detection methods is often employed in labeling experiments

• Pulse-chase experiments are particularly useful for tracking changes in the intracellular location of proteins or the transformation of a metabolite into others over time

Mass Spectrometry Measures the Mass of Proteins and Peptides

• A powerful technique for measuring the mass of molecules such as proteins and peptides is mass spectrometry

  • This technique requires a method for ionizing the sample, usually a mixture of peptides or proteins, accelerating the molecular ions, and then detecting the ions

• In a laser desorption mass spectrometer, the protein sample is mixed with an organic acid and then dried on a metal target

• Energy from a laser ionizes the proteins, and an electric field accelerates the ions down a tube to a detector

Protein Primary Structure Can Be Determined by Chemical Methods and Form Gene Sequences

• The classic method for determining the amino acid sequence of a protein is Edman degradation

  • In the procedure, the free amino group of the N-terminal amino acid of a polypeptide is labeled, and the labeled amino acid is then cleaved from the polypeptide and identified by high-pressure liquid chromatography

• Protein sequences are determined primarily by analysis of genome sequences

• A powerful approach for determining the primary structure of an isolated protein combines mass spectroscopy and the use of sequence databases

Peptides with a Defined Sequence Can Be Synthesized Chemically

• Synthetic peptides that are identical with peptides synthesized in vivo are useful experimental tools in studies of proteins and cells

• Peptides are routinely synthesized in a test tube from monomeric amino acids by condensation reactions that form peptide bonds

Protein Conformation Is Determined by Sophisticated Physical Methods

• X-Ray Crystallography: The use of x-ray crystallography to determine the three-dimensional structures of proteins

  • In this technique, beams of x-rays are passed through a protein crystal in which millions of protein molecules are precisely aligned with one another in a rigid array characteristic of the protein

• Atoms in the crystal scatter the x-rays, which produce a diffraction pattern of discrete spots when they are intercepted by photographic film

• Are extremely complex (composed of as many as 25,000 diffraction spots) for a small protein

• Cryoelectron Microscopy: Although some proteins readily crystallize, obtaining crystals of others—particularly large multisubunit proteins—requires a time-consuming trial-and-error effort to find just the right conditions

  • In this technique, a protein sample is rapidly frozen in liquid helium to preserve its structure and then examined in the frozen, hydrated state in a cryo-electron microscope

• NMR Spectroscopy: The three-dimensional structures of small proteins containing about as many as 200 amino acids can be studied with nuclear magnetic resonance (NMR) spectroscopy

  • In this technique, a concentrated protein solution is placed in a magnetic field and the effects of different radio frequencies on the spin of different atoms are measured

• NMR does not require the crystallization of a protein, a definite advantage, this technique is limited to proteins smaller than about 20 kDa