Chapter 3AMINO ACIDS, PEPTIDES,

AND PROTEINS

中央研究院 生物化學研究所曾湘文 博士Mar. 06, 2007

Lehninger Principles of Biochemistry, Fourth Edition, 2005

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台大莊榮輝老師網頁 : http://juang.bst.ntu.edu.tw/

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3.1 Amino Acids

3.2 Peptides and Proteins

3.3 Working with Proteins

3.4 The Covalent Structure of Proteins

3.5 Protein Sequences and Evolution

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Amino Acids share common structural Features

Proteins are polymers of amino acids, with each amino acid residue joined to its neighbor by a specific type of covalent bond.

The -carbon of AA is a chiral center. Molecules with a chiral center are optically active, they rotate plane-polarized light.

The additional carbons in an R group are designated etc.

Carbon atoms are numbered from one end, giving priority to carbons with substitutions. containing atoms with the highest atomic numbers.

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D, L System (levorotatory vs. dextrorotatory)

Enantiomers -nonsuperimposable mirror images of each other the two forms represent a class of stereoisomers.

The absolute configurations of simple sugars and amino acids are specified by the D, L system.

RS system is used in the systematic nomenclature of organic chemistry and describes more precisely the configuration of molecules with more than one chiral center.

Nearly all biological compounds with a chiral center occur naturally in only one stereoisomeric form, either D or L. The amino acid residues in protein molecules are exclusively L-stereoisomers. D-Amino acid residues have been found only in a few, generally small peptides, including some peptides of bacterial cell walls and certain peptide antibiotics.

6The R groups in this class of amino acids are nonpolar and hydrophobic.

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Nonpolar, Alipatic R Groups

Glycine has the simplest structure, its very small side chain makes no real contribution to hydrophobic interactions

Methionine, one of two sulfur containing amino acids. has a nonpolar thioether group. First AA residue in translation of proteins

Alanine, Valine, Leucine, and Isoleucine could contribute to hydrophobic interaction.

The secondary amino (imino) group of Pro is held in a rigid conformation that reduces the structural flexibility of polypeptide regions containing proline.

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Absorbance of UV by Aromatic Amino acids

All are relatively nonpolar (hydrophobic).

-OH group of throsine can form hydrogen bonds and are important functional group. Can be phosphorylated as well.

All can absorb UV light (280 nm), Tyrosine and Tryptophan are stronger than phenylalanine. Use for protein quantification.

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Polar, Uncharged R Groups

The R groups of these amino acids are more soluble in water, or more hydrophilic, than those of the nonpolar amino acids, because they contain functional groups that form hydrogen bonds with water.

Serine and Threonine has –OH, which contribute to polarity, and could be phosophorelated.

Asparagine and Glutamine are the amides of Aspartate, and Glutamate, and are easily hydrolyzed by acid or base.

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Reversible Formation of Disulfide Bond

Cysteine is readily oxidized to form a covalently linked dimeric AA called cystine (disulfide bond).

The disulfide-linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the structures of many proteins by forming covalent links between parts of a protein molecule or between two different polypeptide chains.

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Positively Charged (Basic) R Groups

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Positively Charged (Basic) R Groups

Lysine has a second primary amino group at position. Its R group has significant positive at pH=7.

Arginine has a positively charged guanidino group Histidine a imidazole group, and is the only standard

amino acid having an ionizable side chain with a pKa near neutrality. It serves as a proton donor/ acceptor in a enzyme-catalyzed reaction

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Negatively Charged (Acidic) R Groups

Two amino acids having R groups with a net negative charge at pH=7 are asparate and glutamate, each of which has a second carboxyl group

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Classify the amino acids by polarity

Juang RH (2003) Biochemistry

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Uncommon Amino Acids - I

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Nonstandard Amino Acids

Some 300 additional amino acids have been found in cells. Are created by modification of standard residues already

incorporated into a peptide. 4-hydroxyproline, a derivative of proline, is found in plant cell

wall protein and collagen; 5-hydroxylysine, derived from lysine, are found in collagen.

6-N Methyllysine is a constituent of myosin. -Carboxyglutamate, found in the blood-clotting protein

prothrombin and Ca2+ binding protein. Desmosine, derivative of four Lys residues, which is found in

the elastin. Selenocysteine is introduced during protein synthesis, and

contains Selenium rather than sulfur of cysteine, derived from serine.

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Uncommon Amino Acids - II

Ornithine and citrulline are not constituents of proteins.

They are key intermediates (metabolites) in the biosynthesis of arginine and in the urea cycle.

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Amino acids Can Act as Acids and Bases

Zwitterion (hybrid ion): dipolar ion, can act as either an acid (proton donor or a base (proton acceptor) - Amphoteric mater: ampholyte (amphoteric electrolytes)

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Amino Acids Have Characteristic Titration Curves

The pKa is a measure of the tendency of a group to give up a proton, with the tendency decreasing tenfold as the pKa increases by on unit.

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Titration Curves Predict the Electric Charge of Amino Acids

Isoelectric point (isoelectric pH): pI, The characteristic pH at which the net electric charge is zero (eg. glycine).

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Titration Curves of Glutamate

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Titration Curves of Histidine

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Effect of the chemical environment on pKa

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Effect of the chemical environment on pKa

The perturbed pKa of glycine is caused by repulsion between the departing proton and the nearby positively charged amino group. The opposite charges on the resulting zwitterion are stabilizing, nudging the equilibrium farther to the right.

The electronegative oxygen atoms in the carboxyl groups, which tend to pull electrons toward them, increasing the amino group to give up a proton.

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3.2 Peptides and Proteins

Formation of a Peptide Bond by Condensation

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A few amino acids are joined - an oligopeptide. Many amino acids are joined, - a polypeptide. “Protein” and “polypeptide” are sometimes used interchangeably, molecules

referred to as polypeptides generally have molecular weights below 10,000 (D), and those called proteins have higher molecular weights.

N terminal C terminal

The pentapeptide serylglycyltyrosylalanylleucine (Ser–Gly–Tyr–Ala–Leu)

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Peptides Can Be Distinguished by Their Ionization Behavior (Alanyl-glutamyl-glycyl-lysine)

The acid-base behavior of a peptide can be predicted from its free -amino and -carboxyl groups as well as the nature and number of its ionizable R groups.

Peptides have characteristic titration curves and a characteristic isoelectric pH (pI) at which they do not move in an electric field.

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Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes Titin, a constituent of vertebrate muscle, which

has 27,000 AAs, and M.W.=3,000,000. Single peptide chain Vs. multisubunit protein:

two or more polypeptide associated noncovalently.

The individual polypeptide chains in a multisubunit

protein may be identical or different. If at least two

are identical the protein is said to be oligomeric, and the identical units (consisting of one or more polypeptide chains) are referred to as protomers.

Ex. Hemoglobin- has four polypeptide subunits: two identical chains and two identical chains, all four held together by noncovalent interactions. Each subunit is paired in an identical way with subunit within the structure of this multisubunit protein, so that hemoglobin can be considered either a tetramer of four polypeptide subunits or a dimer of protomers.

The average M.W. of AA= 110 (128-18)

NutraSweet - artificial sweetener

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Levels of Structure in Protein

Primary: A description of all covalent bonds. The sequence of AA residues Secondary: particularly stable arrangements of AA giving rise to recurring

structural patterns. Tertiary: All aspects of the 3D folding of a polypeptide. Quaternary: The spatial arrangement of multisubunits protein

3.3 Working with Proteins

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Separation and Purification of Proteins

Including size, charge, and binding properties. Crude extract: breaking cells, by osmosis lysis or

homogenization. Fractionation: separate proteins into different fraction

based on size of charge. Salting out: The solubility of proteins is lowered at

high salt concentration. Ammonium sulfate ((NH4)2SO4).

Dialysis is a procedure to separate proteins from solvents

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A Purification Table for a Hypothetical Enzyme

1. Crude cellular extract 2. Precipitation with ammonium sulfate3. Ion-exchange chromatography 4. Size-exclusion chromatography 5. Affinity chromatography

Fraction volume (ml) Total protein (mg) Activity (units) Specific activity (units/mg)

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Protein Purification: Column Chromatography

The expansion of the protein band in the mobile phase is caused by separation of proteins with different properties and by diffusion spreading. As the length of the column increases, the resolution of two types of protein improves.

Rate is decreased and resolution can decline because of the diffusion spreading.

HPLC, or high-performance liquid chromatography. uses high-pressure pumps that

speed the movement of the protein molecules down the column, as well as higher-quality chromatographic materials that can withstand the crushing force of the pressurized flow. By reducing the transit time on the column, - limit diffusional spreading of protein bands and thus greatly improve resolution.

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Ion-exchange Chromatography (net electric charges)

The column matrix is a synthetic polymer containing bound charged groups; those with bound anionic groups (negatively charged) are called cation exchangers,

bound cationic groups (positively charged) are called anion exchangers.

Is effected by pH and salt concentration.

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Size-Exclusion Chromatography (size)

Also called gel filtration chromatography

The column matrix is a cross-linked polymer with pores of selected size.

Larger protein migrate faster than smaller ones because they are too large to enter the pores

41From: 台大莊榮輝上課資料

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Affinity Chromatography (binding specificity) separates proteins by

binding specificities. The proteins retained on

the column are those that bind specifically to a ligand cross-linked to the beads.

After proteins that do not bind to the ligand are washed through the column, the bound protein of particular interest is eluted by a solution containing free ligand.

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High Performance Liquid Chromatography (HPLC)

9010 SolventDelivery System

9050 VariableUV/Vis Detector

HPLC SolventReservoirs

HPLCColumn

RheodyneInjector

9060 Polychrom(Diode Array)

Detector

ComputerWorkstation

Keep an eye onthese 4 screens!

use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution.

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Normal vs. Reversed Phase Chromatography

Normal Phase Reversed Phase

Stationary phase Polar (silica gel) Non-polar (C18)

Mobile phase

Non-polar (organic solvents)

Polar (aqueous/organic)

Sample movement Non-polar fastest Polar fastest

Separation based on

Different polarities (functionality)

Different hydrocarbon content

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Electrophoresis Separation of proteins is

based on the migration of charged protein in an electric field

The migration of a protein in a gel during electrophoresis is a function of its size and shape.

= V / E = Z / f :The electrophoretic mobility V: velocity; E: electrical potentialZ: net chargef: frictional coefficient (shope)

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SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SDS binds to most proteins probably by hydrophobic interaction. One SDS for every two AAs, Thus, each protein has a similar charge-to-mass ratio.

Stains protein: Coomassie blue, Silver, and Sypro Ruby Western blot

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Estimating the Molecular Weight of a Protein

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Isoelectric Focusing

pI of a protein: net charge=0

A pH gradient is established by allowing a mixture of organic acids and bases (ampholytes). Protein migrates until it reaches the pH that matches its pI

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Two-Dimensional Electrophoresis

Separates proteins of identical MW that differ in pI or proteins with similar pI but different MW.

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First dimension: IEF (based on isoelectric point)

SD

S-P

AG

E(based on m

olecular w

eight)

+ -

acidic basic

HighMW

Low MW

Sample

Two-dimensional Gel Electrophoresis

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Silver staining Coomassie blue staining

Sypro Ruby staining

Staining of Polyacrylamide Gels

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Activity vs. Specific Activity Unit: amount of enzyme

causing transformation of 1 mole of substrate per min. at 25 oC under optimal conditions

Activity: Total units of enzyme (U).

Specific activity: Activity of total protein (U/mg)

3.4 The Covalent Structure of Proteins

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The Function of a Protein Depends on Its Amino acid Sequence

Proteins with different function have different AA sequence

Altering primary structure changes the function of proteins

Similar proteins from different species have similar AA sequences.

Function of a protein depends on its structure; Structure depends on sequence.

Polymorphic (polymorphism): AA sequence variation- An estimated 20% to 30% of the proteins are polymorphic in population.

Specific region (Domain)

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Insulins difference by species

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Protein Sequencing (I): Breaking Disulfide Bonds

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Protein SequencingCleaving the Polypeptide Chain – protease

Chymotrypsin

Pepsin

Tyrosine

CNBr

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Protein sequencing (3): Sanger‘s Method vs. Edman degradation

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Cleaving proteins and sequencing and ordering the peptide fragments

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Investigating Proteins with Mass SpectrometryElectrospray ionization mass spectrometry (ESI)

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Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS)

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Trypsin

Peptide mass fingerprinting (PMF) or peptide mappingPeptide mass fingerprinting (PMF) or peptide mapping

Mass Spectrometric Identification of Mass Spectrometric Identification of Proteins - MappingProteins - Mapping

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Obtaining protein sequence information with tandem MS

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How to 0brain a peptide or protein?

purification from tissue, a task often made difficult by the vanishingly low concentrations of some peptides;

genetic engineering Direct chemical synthesis.

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Chemical Synthesis of Peptide (R. Bruce Merrifield)

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Error Rate is Increased as The Polypeptide Chain Gets Longer

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Protein Sequences Can Elucidate the History of Life on Earth For a given protein, the amino acid residues essential for the

activity of the protein are conserved over evolutionary time. The residues that are less important to function may vary over time

If two organisms are closely related, the sequences of their genes and proteins should be similar. The sequences increasingly diverge as the evolutionary distance between two organisms increases.

The members of protein families are called homologous proteins, or homologs - Paralogs (same species) vs. Orthologs (different species)

the rare transfer of a gene or group of genes from one organism to another, a process called lateral gene transfer.

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Protein Homology among Species

Invariant (conserved) residues (same residue) vs. variable residues.

Conservative substitutions: Substitutions with similar amino acid residue (i.e. Arg to Lys).

The number of residues that differ in homologous protein from any two species is in proportion to the phylogenetic (evolutionary) difference between those species.

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Aligning protein sequences with the use of gaps

Within this sequence alignment, a positive score is assigned for each position where the amino acid residues in the two sequences are identical.

In some case, two sequence segments are connected by less related sequences of different lengths - cannot be aligned at the same time – introduce gap (penalties: negative score)

When amino acid substitutions are found within a protein family, many of the differences may be conservative - that is, an amino acid residue is replaced by a residue having similar chemical properties. Ex. Glu – Asp; Leu – Ala.

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Blosum (blocks substitution matrix) table: Blosum62

• The identical residues were given scores based on how often they were replaced, such that amino acids with unique chemical properties (such as Cys and Trp) received higher scores than those more conservatively replaced (such as Asp and Glu).

Higher scores were given to nonidentical residues that occurred frequently than to those that appeared rarely.

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Signature sequences in the EF-1/EF-Tu protein family

Certain segments of a protein sequence may be found in the organisms of one taxonomic group but not in other groups; these segments can be used as signature sequences for the group in which they are found.

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external nodes - Extant species

internal nodes – Extinct ancestor species

Evolutionary tree derived from amino acid sequence comparisons

Electrophoresis

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蛋白質的泳動率 (mobility)

•泳動率與分子上 電荷密度 成正比，而與其分子摩擦力成反比•蛋白質由負極向正極泳動

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蛋白質的帶電性

蛋白質分子上的淨電荷，取決於 環境 pH 高低；若環境 pH 高於其 pI，此蛋白質帶淨負電，反之帶淨正電；若剛好等於其 pI ，淨電荷為零 ( 正電數目等於負電 ) 。同一分子在不同 pH 環境下，可能帶不同 淨電荷 。

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膠体電泳

組成膠体的分子長鏈間，有相當大的空間，可降低與蛋白質間的摩擦力，且可增大樣本体積，適用於巨分子電泳，如核酸及蛋白質。

澱粉膠体電泳 (starch gel electrophoresis) 聚丙烯醯胺電泳 (polyacrylamide gel

electrophoresis, PAGE) 洋菜糖膠体電泳 (agarose gel electrophoresis)

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聚丙烯醯胺膠体電泳 (PAGE)

PAGE 是最普遍的蛋白質電泳方式。 原態膠体電泳 (native-PAGE) 及活性分析

蛋白質以原態進行電泳，因此酵素活性在電泳後得以保持，可在膠片上直接做活性測定或染色。因為樣本蛋白質保持在原態下，所帶的電荷、分子大小、分子形狀等，對其泳動率均有影響，與下述 SDS-PAGE 不同。

SDS 膠体電泳及分子量測定 SDS (sodium dodecyl sulfate) 是界面活性劑，可使蛋白質變性，並在分子表面均勻佈上一層負電荷。 因此在 SDS-PAGE 系統中，樣本分子的泳動率，僅取決於其分子量，而與原來分子所帶的電荷無關，故 SDS-PAGE 可用來測定變性狀態 (denatured) 蛋白質之 分子量，與原態 (native) 分子量可能不一樣。

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PAGE 膠体的組成 單体分子 (monomer)

丙烯醯胺 (acrylamide) ， H2C=CH-CO-NH2 。 Acrylamide 及下面的 Bis 都有神經毒性，要帶手套，並避免吸入塵埃。

架橋分子 (bridge) Bis [N,N'-methylene-bis(acrylamide)] 可看作兩個丙烯醯胺單体分子連結在一起，可形成分叉點，以構成立体結構。

自由基 (free radical) 產生者 通常使用 過硫酸銨 (ammonium persulfate, APS) 或者 riboflavin ( 即 維生素 B2) 。

催化劑 TEMED (tetramethylethylenediamine) 幫助游基電子的傳遞。

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鑄膠反應

自由基形成生成自由基，使單体分子成為自由基型式。

聚合反應自由基單体可首尾相接，以連鎖反應形成大分子的長鏈。

交錯連結若架橋分子加入聚合反應，則形成網狀三次元結構。

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電泳的焦集作用