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本课件为英文教材第三章，围绕蛋白质的结构层次展开：先讲氢键、疏水作用等影响结构的作用力，再讲一级结构序列的测定与比对，随后系统讲二级结构、蛋白质折叠与三级结构、亚基相互作用与四级结构，最后讲蛋白质纯化技术的一般流程与常用方法。
Chapter 3 Protein Structures
Proteins are a diverse and abundant class of biomolecules, constituting more than 50% of the dry weight of cells. This diversity and abundance reflect the central role of proteins in virtually all aspects of cell structure and function. An extraordinary diversity of cellular activity is possible only because of the versatility inherent in proteins, each of which is specifically tailored to its biological role.
Definitions of Protein StructurePrimary structure –amino acid sequenceSecondary structure–local conformation of peptide backboneTertiary structure–interaction of different secondary structures on same polypeptideQuaternary structure–interaction of different polypeptides in structure that has more than one protein component
Primary Secondary Tertiary Quaternary
3.1 • Forces Influencing Protein Structure Hydrogen Bonds Hydrophobic Interactions Electrostatic Interactions Van der Waals Interaction
Non-bonding Forces Influencing Protein StructuresAmino acids of a protein are joined by covalent bondinginteractions. The polypeptide is folded in three dimension by non-bondinginteractions. These interactions can easily be disrupted by extreme pH, temperature, denaturants, reducing reagents. We will discuss the nature of these types of forcesH-bond interactions (12-30 kJ/mol)Hydrophobic Interactions (<40 kJ/mol)Electrostatic Interactions (20 kJ/mol)Van Der Waals Interactions (0.4-4 kJ/mol)The total inter-atomic force acting between two atoms is the sumof all the forces they exert on each other.
H-bond describes a favorable interaction between a proton bonded to an electronegative atom and an atom carrying a lone pair of electrons:D-H + A D H AAcceptors (A):Donors (D):This interaction is very important for maintaining protein backbone interactionsHydrogen bonds
Hydrogen bondsThis figure shows hydrogen bonds(white dash lines) are important to hold a small in place at the active site of an enzyme.H-bonds in helixPeptide bond
Hydrophobic InteractionsHydrophobic interactions minimizes interactions of non-polar residues with solvent. Thus nonpolar regions of biological macromolcules are often buried in the molecules interior to exclude them from the aqueous milieu. However non-polar residues can also be found on the surface of a protein. They may participate protein-protein interactions.This type of interaction is entropy driven.
Electrostatic InteractionsCharged side chains in protein can interact favorably with an opposing charge of another side chain according to Coulomb’s law:Examples of favorable electrostatic interaction include that between positively charged Lys and negatively charged Glu. Salts have the ability to shield electrostatic interactions.
Examples of Electrostatic InteractionsIntramolecular ionic bonds between charged amino acid residues in a protein:Magnesium ATP
van der Waals Interactionsvan der Waals interaction between two atoms is a result of electron charge distributions of the two atoms. For atoms that have permanent dipoles:Dipole-dipole interactions (potential energy ~r-3)Dipole-induced dipole interactions (potential energy ~r-5)For atoms that have no permanent dipoles:Transient charge distribution induces complementary charge distribution (also called dispersionor London dispersion force) (potential energy ~r-6)Repulsionbetween two atoms when they approach each other due tooverlapping of electron clouds (potential energy ~r-12)d+d-d-d+d+d-transientdipoletransientdipole
van der Waals Interactions (Cont.)In general, the permanent dipole contribution are much less that the dispersion and repulsion forces. Thus the van der Waals potential can be expressed as 1/r12-1/r6. The characteristics of van der Waals potential is shown below: r0is the sum of van der Waals radii for the two atoms. Van der Waals forces are attractive forces when r> r0and repulsive when r< r0.rVan der Waals potentialVan der Waals Forcer0r0Van der waals redii of common atoms (nm):H 0.1 nm C 0.17 nmN 0.15 nmO 0.14 nmP 0.19 nmS 0.185 nm
3.2 • Sequence(primary structure) in Protein StructureAll of the information necessary for folding the peptide chain into its “native” structure is contained in theamino acid sequence of the peptide.
Proteins are linear polymers of amino acidsTerminology:Residues amino acids in proteinsDipeptide a peptide containing two amino acidsPolypeptide a peptide containing more than a dozen amino acidsMonomer a protein containing a single polypeptideMultimers a protein containing more than one polypeptideHomodimer a protein contain two identical polypeptideHeterodimers a protein contain two different polypeptideTetramers a protein containing four polypeptides (can be homo-or hetero-) For a polypeptide of N amino acids, there is 20Npossible amino acid sequences. Thus there is virtually unlimited number of amino acid sequencesNH2COOHAmino acids
Peptide bonds connect amino acids into linear un-branched chainsFormed by a condensation reaction between the -NH2(amido) group of one amino acid and the –COOH(carboxyl) group of another.
Protein Sequence (Primary structure)The amino acid sequence of a proteinis an unique characteristic of the proteinis encoded by the nucleotide sequence of DNA, is thus a form of genetic informationis read from the amino terminus to the carboxyl terminus
Structure of peptide bondTwo amino acids are joined by the peptide bond, a reaction catalyzed by the enzyme called ribosome in all cells:Due to the double bond character, the six atoms of the peptide bond group are always planar!
Analysis of Protein Sequences Proteins can be sequenced in two ways:real amino acid sequencingsequencing the corresponding DNA in the geneAmino Acid Analysis of Proteins6mol/L HCl @ 110°C for 24, 48, 72 hours in sealed glass vials (Trp is destroyed, must be analyzed by other means)Each reaction mixture is loaded on an ion-exchange column where each amino acid can be eluted according to their chargeEluted amino acids are quantified by reacting to ninhydrin (postcolumn derivatization)This method has been automated (amino acid analyzer) and allows analysis of only the amount of each type of amino acids, but gives no information on the order of amino acids nor the absolute amount of each amino acid.
Edman Degradation MethodEdman degradation uses Edman reagent to determine the order of amino acids from the N-terminal end of a protein.
CNBr CleavageCNBr(cyanogen bromize) acts only on methionine residuesCNBr is useful because proteins usually have only a few Met residues
Cleavage of Disulfide BondsOxidation of a disulfide by performic acid:Reduction of S-S bridges by sulfhydryl compound:
Protein Sequencing ProcedureIf more than one polypeptide chain, separate.Cleave (reduce) disulfide bridgesDetermine composition of each chain amino acid analysis;Determine N-and C-terminal residues by Edman degradation and C-terminal proteasesCleave each chain into smaller fragments by site-specific proteases and determine the sequence of each chain by the above methodsRepeat the above step, using a different cleavage procedure to generate a different set of fragments.Reconstruct the sequence of the protein from the sequences of overlapping fragments
Mass SpectrometryMass spectrometry is increasingly used in protein identificationMS determines the mass-to-charge (m/z) ratio of protein mixtures or peptides (if hydrolyzed by a protease):Evaporate and ionize molecules in a vacuumSeparate the ions in space and/or time based on their m/z ratios;Measure different m/z ratios
Mass SpectrometerSchematic drawing of a mass spectrometer. (1) Highly charged protein droplets are formed by electrostatic dispersion of a protein solution through a glass capillary subjected to a high electric field (electrospray); This step can also be replaced by matrix-assisted desporption ionization (MALDI) (2) Protein droplets are accelerated by electrostatic forces in vacuum; (3) the time it takes for a protein to arrive at the mass spectrometer depends on its m/z. Thus an unknown protein mass can be obtained by comparing its m/z with that of a standard protein.Mass analyzer++++++++++(1)(2)(3)High voltageVacuum
3.3 • Secondary Structure in Proteins Any discussion of protein folding and structure must begin with the peptide bond, the fundamental structural unit in all proteins.
The partial double bond character of the peptide bond.
Bond distances support double bonded nature of peptide bond
Trans configuration of peptide bond is favored for all amino acids except proline
Structure of the peptide bond (M5.12)Notice the planar nature of the peptide bond. The trans form is favored. Delocalization of the -electron orbital over the O-C-N accounts for the partial double bond character of the peptide bond.
Although the peptide bond is planar, there is rotation around the bonds to the alpha carbon of each amino acid residue
Rotation is allowed only on both sided of the a-carbon. The angles of rotation are defined as (phi) and (psi)The structure of the protein is defined by the rotation around the polypeptide chain (M6.2)
The amide or peptide bond planes are joined by the tetrahedral bonds of the a-carbon. The rotation parameters are fand y. The conformation shown corresponds to f= 180°and y= 180°. Note that positive values of fand ycorrespond to clockwise rotation as viewed from Ca. Starting from 0°, a rotation of 180°in the clockwise direction (+180°) is equivalent to a rotation of 180°in the counterclockwise direction (-180°).
Most of combination of fand y are sterically forbidden.
G. N. Ramachandran and his coworkers in Madras, India, first showed that it was convenient to plot fvalues against yvalues to show the distribution of allowed values in a protein or in a family of proteins.A typical Ramachandran plotis shown in this figure.
Pauling principles for proteins structure(1950)1.The bond lengths and angles of the amino acids and peptides in proteins should be as close as possible to those in isolated amino acids and peptides. 2.No two atoms should approach each other closer than allowed by their van der Walls radii.3.The amide bond should remain planar and in the trans configuration. Rotation is possible on both sides of the carbon. 4.Noncovalent bonding is essential for maintaining structure and hydrogen bonding between amide protons and carbonyl oxygens are the most obvious.
Secondary structure: -helix
Although helices are uncommon in manmade architecture, they are a common structural theme in biological macromolecules—proteins, nucleic acids, and even polysaccharides.
Secondary structure: the helix
A hydrogen bond between the amide proton and carbonyl oxygen of adjacent peptide
Dipole moment of a helix (L6-6)
The ahelix
Right and left handed helices (L box 6-1)
Interaction between R groups 3 residues apart in an helix (L6-5)
Other Helical Structures
Secondary structure: -sheet
Secondary structure: the beta sheet
parallel b-pleated sheet antiparallel b-pleated sheet
b sheets are depicted by arrows in protein structure schematic diagrams
bsheets often fold back upon themselves to form bbarrels
Secondary structures: Turns
Beta turns (M6.18)
turn (M6.19)
Proline isomers (L6-8)
Loops between alpha helices or beta sheets often are very important functional domains of proteins
Secondary structures: The Beta-Bulge
3.4 • Protein Folding and Tertiary Structure When the polypeptide chains of protein molecules bend and fold in order to assume a more compact three-dimensional shape, a tertiary level of structureis generated. It is by virtue of their tertiary structure that proteins adopt a globular shape. A globular conformation gives the lowest surface-to-volume ratio, minimizing interaction of the protein with the surrounding environment.
The folding of a single polypeptide chain in three-dimensional space is referred to as its tertiary structure. Why proteins tend to form tertiary structures?
First,secondary structures—helices and sheets—form whenever possible as a consequence of the formation of large numbers of hydrogen bonds.Second,a-helices and b-sheets often associate and pack close together in the protein. No protein is stable as a single-layer structure. Third,because the peptide segments between secondary structures in the protein tend to be short and direct, the peptide does not execute complicated twists and knots as it moves from one region of a secondary structure to another.
that proteins can be grouped into three large classes based on their structure and solubility:fibrous proteins,globular proteins,and membrane proteins.
Fibrous proteinscontain polypeptide chains organized approximately parallel along a single axis, producing long fibers or large sheets. Such proteins tend to be mechanically strong and resistant to solubilization in water and dilute salt solutions. Fibrous proteins often play a structural role in nature. Fibrous proteins tend to have relatively simple, regular linear structures. These proteins often serve structural roles in cells. Typically, they are insoluble in water or in dilute salt solutions.
globular proteinsare roughly spherical in shape. The polypeptide chain is compactly folded so that hydrophobic amino acid side chains are in the interior of the molecule and the hydrophilic side chains are on the outside exposed to the solvent, water. Consequently, globular proteins are usually very soluble in aqueous solutions. Most soluble proteins of the cell, such as the cytosolic enzymes, are globular in shape.
Membrane proteinsare found in association with the various membrane systems of cells. For interaction with the nonpolar phase within membranes, membrane proteins have hydrophobic amino acid side chains oriented outward. As such, membrane proteins are insoluble in aqueous solutions but can be solubilized in solutions of detergents. Membrane proteins characteristically have fewer hydrophilic amino acids than cytosolic proteins.
a-Keratin Fibroin and b-Keratin: b-Sheet Proteins Collagen: A Triple Helix Fibrous proteins
The structure of α -keratins
Permanent waving is biochemical engineering
Fibroin and b-Keratin: b-Sheet ProteinsThe fibroinproteins found in silk fibers represent another type of fibrous protein. These are composed of stacked antiparallel b-sheets. In the polypeptide sequence of silk proteins, there are large stretches in which every other residue is a glycine.
As previously mentioned, the residues of a b-sheet extend alternately above and below the plane of the sheet. As a result, the glycines all end up on one side of the sheet and the other residues (mainly alanines and serines) compose the opposite surface of the sheet. Pairs of b-sheets can then pack snugly(紧贴身地) together (glycine surface to glycine surface or alanine—serine surface to alanine—serine surface).
The structure of silk fibroin (M6.12)
β -keratin-(-Gly-Ala-)n-
silk fibroin
The b-keratins found in bird feathers are also made up of stacked b-sheets.
Collagen is the matrix protein of bones, tendons (腱) and skin.
The basic unit is tropocollagen, a left handed triplehelix. .Tropocollagen has a molecular weight of 285,000 and consists of three intertwined polypeptide chains, each about 1000 amino acids in length. Tropocollagen molecules are about 300 nm long and only about 1.4 nm in diameter. Several kinds of collagen have been identified.
There are three types of collagenType I collagen, which is the most common, consists of two identical peptide chains designated a1(I) and one different chain designated a2(I). Type I collagen predominates in bones, tendons, and skin. Type II collagen, found in cartilage, and type III collagen,found in blood vessels, consist of three identical polypeptide chains.
The triple helix of collagen is very unique with Gly-Pro-Pro/HyProevery 3ed residue (Gly in the center) and 3.3 residues per turn.
Hydroxylation of Pro is very common (for structural purpose) and of lysine less (for attachment of sugars). Hydroxylation depends on Vitamin C . Vitamin C deficiency causes scurvy (坏血病).
Gly , because its small size, is required at the tight junction where the three chains are in contact.
The structure of collagen fibers (M6.13)
Crosslink 1
DesmosineCrosslink 2
Bone consists of microcrystals of hydroxyapatite,Ca5(PO4)3OH, embedded in a matrix of collagen fibrils. When new bone tissue forms, the formation of new hydroxyapatite crystals occurs at intervals of 68 nm. The hole regions of collagen fibrils may be the sites of nucleation for the mineralization of bone.
Globular ProteinsFibrous proteins, although interesting for their structural properties, represent only a small percentage of the proteins found in nature. Globular proteins,so named for their approximately spherical shape, are far more numerous.
Tertiary Structure: the 3-d arrangement of all amino acidsDescribes the number, size and arrangement of its secondary structuresStabilized by hydrophobic interactions between non-polar side-chains and, in some proteins, by disulphide bonds.Monomeric proteins (single polypeptide chain) -tertiary structure is the highest level of organisation.
Globular protein structures are compact and variedHuman serum albumin(Mr 64500)
Structural Diversity Reflects Functional Diversity in Globular ProteinsIn a globular protein, different segments of a polypeptide chain (or multiple polypeptide chains) fold back on each other.The folding also provides the structural diversity necessary for proteins to carry out a wide array of biological functions. Globular proteins include enzymes, transport protein, motor proteins, regulatory proteins, immunoglobulins, and proteins with many other functions.
Helices and sheets in globular proteins
Packing ConsiderationSecondary structures pack closely to one another and also intercalate（插入）with (insert between) extended polypeptide chains. There usually are spaces between secondary structure. Nearly all of this spaces is in the form of very small cavities. Cavities the size of water molecules or larger do occasionally occur, but they make up only a small fraction of the total protein volume.
Ordered, Nonrepetitive Structure the segments of the polypeptide chain that cannot be classified as defined secondary structures, such as helices or sheets, have been traditionally referred to as coilor random coil. Both these terms are misleading. Most of these segments are neither coiled nor random, in any sense of the words. These structures are every bit as highly organized and stable as the defined secondary structures. They are just more variable and difficult to describe.
Different graphical representations of the same protein
Flexible, Disordered Segments In addition to nonrepetitive but well-defined structures, which exist in all proteins, genuinely disordered segments of polypeptide sequence also occur.
Flexible, Disordered Segments
Motion in Globular Proteins These motions should be viewed as momentary oscillations(震动)about a single, highly stable conformation. Proteins are thus best viewed as dynamic structures.The allowed motions may be motions of individual atoms, groups of atoms, or even whole sections of the protein. Furthermore, they may arise from either thermal energy or specific, triggered conformational changes in the protein.
A class of slower motions, which may extend over larger distances, is collective motions.These are movements of groups of atoms covalently linked in such a way that the group moves as a unit. Such groups range in size from a few atoms to hundreds of atoms.
Conformational changesinvolve motions of groups of atoms (individual side chains, for example) or even whole sections of proteins.
Forces driving the Folding of Globular ProteinsFolding of polypeptides is subject to an array of physical and chemical constraints.So there are some basic rules in domain
1.Hydrophobic interactionsHydrophobic interactions make a large contribution to the stability of protein structures. Burial of hydrophobic amino acid R groups so as to exclude water requires at least two layers of secondary structure.
2. αhelices and βsheets interactionsWhere they occur together in proteins, αhelices and βsheets generally are found in different structural layers. This is because the backbone of a polypeptide segment in the βconformation cannot readily hydrogen bond to an a helix aligned with it.
3. Adjacent residues interactions Polypeptide segments adjacent to each other in the primary sequence are usually stacked adjacent to each other in the folded structure. Although distant segments of a polypeptide may come together in the tertiary structure, this is not the norm.
4. Relative rigidity of elements of secondary Connections between elements of secondary structure cannot cross or form knots.
5.βconformation tends to be twisted. The βconformation is most stable whenthe individual segments are twistedslightly in a right-handed sense.Thisinfluences both the arrangement of βsheets relative to one another and the path of the polypeptide connection between them.
Two arrangements of βstrands stabilized by the tendency of the strands to twist
Globular proteins have a variety structuresCyt c
lysozyme
ribonuclease
Classification of globular proteinsIt is important to note that the similarities of tertiary structure within these groups do not necessarily reflect similar or even related functions. Instead, functional homologyusually depends on structural similarities on a smaller and more intimate scale.
As rapidly development of biochemistry, the number of known three-dimensional protein structures is in the thousands and more than doubles every two years. This wealth of structural information is revolutionizing our understanding of protein structure, the relation of structure to function, and even the evolutionary paths by which proteins arrived at their present state, which can be glimpsed in the family resemblances among proteins that are revealed as protein databases are sifted(详审) and sorted.
Vast amount of information available over the Internet from resources such as the Protein Data Bank (PDB), an archive of experimentally determined three-dimensional structures of biological macromolecules,
The sheer variety of structures can seem daunting, yet as new protein structures become available it is becoming increasingly clear that they are manifestations of a finite set ofrecognizable, stable foldingpatterns.
Analysis of many globular proteins reveals common structure patterns
Supersecondary structure, also called motifsor simply folds, are particular stable arrangements of several elements of secondary structure and the connections between them. Those three terms are often used interchangably
Motifs are regular combinations of secondary structuresA coiled coil motif is formed by two or more helices wound around one another
Helix-loop-helix motifHas certain hydrophilic residues at invariant positions in the loop. O atoms in these residues bind a Ca 2+ion through H-bonds.
Zinc-finger motifThree secondary structures (an -helix and 2 -strands with an anti-parallel arrangement) form a finger-like bundle held together by a zinc ion.Often found in DNA-and RNA-binding proteins.
Two arrangements of βstrands stabilized by the tendency of the strands to twist
Constructing large motifsfrom smaller ones
Polypeptides with more than a few hundred amino acid residues often fold into two or more stable, globular units called domains. In many cases, a domain from a large protein will retain its correct three-dimensional structure even when it is separated (for example, by proteolytic cleavage) from the remainder of the polypeptide chain.
A protein with multiple domains may appear to have a distinct outline for each domain,but, more commonly, extensive contacts between domains make individual domains hard to discern. Different domains often have distinct functions,such as the binding of small molecules or interaction with other proteins. Small proteins usually have only one domain (the domain is the protein).
Typical domain structures of globular proteins
Proteins with significant primary sequence similarity are said to be in the same protein family;•Two or more families with little primary sequence similarity sometimes make use of the same major structural motif and have functional similarities; these families are grouped as superfamilies.
The Structural Classification of Protein(SCOP) database:all α allβ α/β α+β
Click here
Important website for protein structure HHH//www.ncbi.gov/HHH//www.ebi.au.uk/HHH//www.rcsb.org/
How Do Proteins Know How to Fold? Christian Anfinsen’s experiments demonstrated that proteins can fold reversibly.
Thethermodynamic ofprotein foldingdepicted as free-energy funnel
A classical example isthe denaturation andrenaturation ofribonuclease(Anfinsen experiment)
The possibilities ofcorrect disulfide bondsFirst 7Second 5Third 3Fouth 1Total 7Х5 Х3=105
Isomers of transand cis of peptide bondsAlso there are uncountableconfiguration of dihedral angles.It is impossible that the folding of protein only just by chance.
Molecular Chaperones: Molecular Chaperones are proteins that fold globular protein
Some proteins undergoassisted foldingMolecular chaperone:Hsp70(Mr near 70k, dnaK anddnaJ in E.coli.)Chaperonins(hsp60s orcpn60s, one of them is GroELand GroES in E.coli.)
Fig. 6-13B and T
Large conformational changes coupled to folding•~ 70 kDa limit
Structure of a chaperonin (M6.26)
Assisted folding ofproteins1.Aggregation is prevented and folding is assisted by Hsp70 proteins, DnaJ and DnaK. 2.Chaperonins, GroEL/GroES actively fold proteins by repeated cycles of binding and movements3.Protein disulfide isomerases (PDI) help to form the correct s-s bridges.4.Peptidyl proline isomerase (PPI) helps to return the prolines to the correct trans (in most cases) form.
Death by misfolding:the prion disease(madcow disease, PrPmisfolding)A stained section of the cerebralcortex from a patient showsspongiform degeneration
3.5 • Subunit Interactions and Quaternary StructureProtein Quaternary Structures Range from Simple Dimers to Large Complexes
Quaternary Structure: the number and positions of sub-units in a multimeric proteinMultimeric proteins: two or more polypeptide chains or sub-units held together by non-covalent bonds.Haemagglutinin is a trimer of 3 identical subunits.
A multisubunit protein is also referred to as a multimer. Multimeric proteins can have from two to hundreds of subunits. A multimer with just a few subunits is often called an oligomer.
Symmetry of quaternary structure If a multimer is composed of a number of nonidentical subunits, the overall structure of the protein can be asymmetricand quitecomplicated.However, most multimers have identical subunits or repeating groups of nonidentical subunits, usually in symmetric arrangements.The repeating structural unit in such a multimeric protein, whether it is a single subunit or a group of subunits, is called a protomer(原体）
The quaternary structure of deoxyhemoglobin（脱氧血红蛋白）(a rebbon representation)
The quaternary structure ofdeoxyhemoglobin(a space-filling model)
Oligomers can have either rotation symmetryor helical symmetry
Dihedral(两面角)
Icosahedral(20面角)
Viral Capsids. (a) Poliovirus(脊髓灰质炎病毒)There are limits to the size ofprotein
Viral Capsids（衣壳）. (b) Tobacco mosaic virus
Forces Driving Quaternary Association The favorable interactions include van der Waals interactions, hydrogen bonds, ionic bonds, and hydrophobic interactions.
Modes and Models for Quaternary Structures
Tubulin（微管蛋白）
Structural and Functional Advantages of Quaternary AssociationStability One general benefit of subunit association is a favorable reduction of the protein’s surface-to-volume ratio. The surface-to-volume ratio becomes smaller as the radius of any particle or object becomes larger.
Genetic Economy and Efficiency Less DNA is required to code for a monomer that assembles into a homomultimer than for a large polypeptide of the same molecular mass.
Cooperativity Most oligomeric enzymes regulate catalytic activity by means of subunit interactions, which may give rise to cooperative phenomena.
3.6 Protein Purification techniqueThe more the specific assay, the more effective the purificationThe easier the assay, the more rapid the purification
Differential centrifugation as a first step in protein purification
Protein Purification Techniques –Large ScaleSalting outDialysisGel-Filtration ChromatographyIon-Exchange ChromatographyAffinity ChromatographyHigh-Pressure Liquid Chromatography
Dialysis –Separation based on sizeExtra usages:Reverse dialysisUltrafiltration
Gel-Filtration Chromatography –Separation based on sizeSephadax Sepharose
Ion-Exchange Chromatography –Separation based on charge
Types of ion-exchange chromatography media
Affinity ChromatographySmall molecules are attached to beads and complex protein mixtures are applied. Bound proteins can be eluted with the small molecule or with denaturing reagents (urea, guanidine, etc.)
High Pressure Liquid ChromatographyHigh pressure limits diffusion and increases interactions with chromatography mediaHPLC gives very high resolution of protein components
Polyacrylamide Gel Electrophoresis (PAGE)Molecules are separated by size in an electric fieldSmaller molecules migrate more rapidly through porous gel matrix
Polyacrylamide is a useful polymer for gel electrophoresis because both the concentration and cross-linking of the gel can be controlledAcrylamide Polymerization
Polyacrylamide(聚丙烯酰胺）gel electrophoresis（PAGE）can occur under both denaturing and reducing conditions
Proteins are detected in PAGE gels by staining or autoradiography
Isoelectric Focusing relies on the migration of charged proteins in an electric field
Isoelectric focusingEvery protein has an isoelectric point (pI). This is the pH at which the protein has no electrical charge.A protein with many basic amino acids (Arg, Lys, His) will have a basic pI greater than 7 and a protein with acid residues (Glu, Asp) will have an acidic pI less than 7.Stable gradients of pH in gels can be created using polyampholytes and proteins loaded on these gels will migrate to the pH corresponding to their pI.
Isoelectric focusing is often combined with SDS-PAGE to generate two dimensional gels.
Protein Purification SchemesCan be used to compare different methodologies for purification of the same proteinCan be used to standardize the purification of proteins in different laboratories
Zonal Density Gradient CentrifugationMolecules are separated by the rate at which they sediment in a density gradient
The sedimentation coefficient is a means of quantifying density sedimentation rates
Hydrophobic Interaction Chromatography (HIC)Hydrophobic groups are covalently linked to Support matrix. Phenyl Sepharos®In the presence of high salt concentrations, proteins bind to the phenyl groups by virtue of hydrophobic interactions. Proteins in a mixture can be differentially eluted from the phenyl groups by lowering the salt concentration or by adding solvents such as polyethylene glycol to the elution fluid.
Mass Spectrometry can accurately determine the mass of proteins
The Time of Flight of the protein ions is related to the mass to charge ratio of the protein ion
AbundantredundantVirtuallyInherentVersatilityTailor toSensorQuaternary Rigiditysumof Exert ReagentfavorableBackboneMilieuInteriorAqueousEntropyParticipateElectrostaticIntramolecularTransientDispersionRepulsionRadiiNew WordsDelocalization OrbitalAccounts for PlanarRotationCapillaryVacuumAcceleratedStericalValuesDefaultSpiral
AlignmentBulgeCylinderAmphipathicOppositeDepictDiagramBarrelLaterallyOrientation
The end of chapter 3
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