Amino Acids And Proteins

AMINO ACIDS PROTEINS  Most abundant macromolecules living in cells  Great variety  Great diversity in biological function  Molecular instruments by which genetic information is expressed  Gk protos : first or foremost  All are constructed from same ubiquitous 20 AA  The 20 AA are joined in many different combinations and sequences  Producing thousands of different structures, different properties and activities  Different products and enzymes, hormones, antibodies, lens, feathers, horns

>solubility in water -the identity/distinctive >size, structure  helps to dictate the folding of the protein Alpha Carbon atom  A hydrogen atom  Amino group (hence “amino” acid) -the amino group (-NH2) accepts a proton and becomes (-NH3+)  Carboxyl group (-COOH) -this gives up a proton and is thus an acid (hence amino “acid”) -becomes dissociated (-COO-)  R group  

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 AMINO ACIDS  Primary amines  Contain an amino group (NH3) group connected to an alpha carbon and to which other substituents are attached  carboxyl group (COOH)  Variable side chain R  H  A-amino acids because they have a primary amino group (-NH2) and a carboxylic acid group (-COOH) attached to the alpha carbon  Amino acid common structure consists of a central C atom which is bonded to a: -amino (-NH2) - carboxyl group (-COOH) -Hydrogen atom (-H) -Side chain group (-R)  Variable side group (-R)  determines: -its special properties -size, shape, reactivity >electric charge

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No asymmetric carbon: GLYCINE is not asymmetric Alpha carbon is asymmetric (except GLYCINE) chiral center -tetrahedral bonding around the alpha C The 4 groups can occupy 2 different arrangements in space, enantiomers or stereoisomers (nonsuperimposable mirror images) Optically active 2 different arrangements of the groups around the alpha carbon: 1. Enantiomers-mirror image 2. Stereoisomers Non-superimposable AA are L or D depending on the position of the amino group Regular tetrahedron. It is symmetrical and a mirror image is no different from the original The mirror images is not identical The mirror image cannot be superimposed on the original

activity of the AA itself but rather to the optical activity of the isomer of glyceraldehyde from which that AA can theoretically be synthesized (Dglyceraldehyde is dextrorotary, Lglyceraldehyde is levorotary) 

Threonine and Isoleucine have 2 chiral carbons each, thus producing 4 possible stereoisomers each

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L forms: -humans and most foods are made up almost exclusively of L form aminos D form proteins are produced by exotic sea-dwelling organisms such as cone snails -abundant components of the peptidoglycan cell walls of bacteria -D-serine may act as a neurotransmitter in the brain

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D-AA that occur naturally -Free: D serine, D asp (brain tissue) -D ala, D glu (cell wall of g+ bacteria) -In peptides and antibiotics produced by bacteria, fungi, reptiles

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imidazole ring of histidine is aromatic at all pH values unprotonated imidazole is nucleophilic (imidazole) and can serve as a general base, while the protonated form (imidazolium) can serve as a general acid acid form, the imidazolium ion (B), is a resonance hybrid imidazole Conjugate base (A or C) Either of the two ring nitrogens can release a proton (H+) to produce the conjugate base form

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Glycine (Gly, G) is the simplest and smallest of all AA -the only one which is not optically active since it has a single H atom as its side chain

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Proline in which the R group makes up part of a ring which also includes the amino group and the alpha carbon atom.

“handedness” is called chirality and the mirror images are called

enantiomers

If this compound is chiral and is present as just one of the enantiomers (mirror images), the plane of the polarized light will be rotated The L and D convention for AA configuration refers not to the optical

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Since the amino group in proline is involved in two carbon-nitrogen bonds, it is a secondary amino group

Properties  Solubility: -generally soluble in water and insoluble in non-polar organic solvents such as hydrocarbons > Charged functional group: readily soluble in polar solvents (H2O, ethanol); insoluble in non-polar solvents ( benzene, ether)  Colorless -Aromatic AA absorb UV  MP -amino acids are crystalline solids with high melting points  Optical activity -With chiral center: optically active  Ultraviolet absorption -Tyr, Phe, Trp : absorb high wavelength UV > Try absorbs light at 280 nm > Absorption bands arise from interaction of radiation with electrons on the aromatic rings  Acid – base -Amino and COOH groups -pKa  pKa COOH : ~2.2 Amino: ~9.4  At pH 7 the amino is protonated NH3+ and carboxyl is in COO(conjugate base or carboxylate form)  AA with single amino and single carboxyl group will have net charge of zero : Zwitterion; which are electrically neutral  In an electric field : stationary Zwitterions - comes from the German word “zwitter” meaning “hybrid” - protonated ammonium group with a (+) charge - deprotonated carboxylate groups with a (-) charge - Net charge: NEUTRAL - At certain compound specific pH: Isoelectric point (pI)

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Isoelectric point (pI) - pH at which AA has no net charge (does not move in electric field) -the ph at midway between the pK values on either side of the Zwitterion specie pI of neutral AA ~6 -Acidic AA (very much<6) -Basic AA (very much >6) Physical properties are influenced by ionic state of COOH and NH2 and other ionizable group in the R Each AA has either 2 or 3 pKa values and these differ among different AA At different pH AA have different net charges AA have both an amine and carbocylic acid functional groups are therefore both acid and base at the same time Zwitterions have minimal solubility at their isoelectric point and AA are often isolated by precipitation from water after adjusting the pH to their isoelectric point -no mobility in electrophoresis

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At pH > 2.2: COOpredominates At pH between 2.2-9.4 the predominant state of the molecule: COO- and NH3+ -zwitterions Can be in 3 general forms: 1. Protonated form (dipolar with charge): -they can dissociate -NH3+ and COO2. Unprotonated/ Neutral form: -they cannot dissociate -NH2 and COOH 3. Amino group: ~9.6 pH>NH3+ Carboxyl group: __________ At low pH the amino and carboxyl groups will be protonated and the molecules will be in the acid form COOH: ~2.2 As the pH is increased (beyond 2.2, COOH dissociates) towards neutrality, the AA become zwitterions having both negative and positive charges AS the pH increase further (beyond pK of aa) NH3 dissociates

Amino acid in solution at isoelectric pH are mainly dipolar ions This is generally how AA exist at cellular pH Because of their amino and carboxyl groups, proteins in solution resist changes in acidity and alkalinity and so are important biological buffers Charged R (basic and acidic) stabilize protein conformations thru ionic interactions or salt bridges

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Dissociation constant amino group and the carboxyl group of the amino acid have PKs of about 2.3 and 9.6 respectively can exist in three general forms  PKs of AA - ~9.6: amino group (mean for all AA)  At pH < than this: NH3+ - ~2.3 carboxyl group (mean for the 20 AA: ~ 2.2)

Reactions:  Between AA and carboxyl groups -formation of peptide bonds  Peptide bond -condensation reaction that is through dehydration synthesis that releases water and the new “AA residue” held together by a peptide bond -partially double bond character of the peptide bond The O, C, N and H atoms are nearly planar and there is no rotation about the peptide bond

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20 proteinogenic AA 21st amino acid Selenocysteine -Found in some proteins > Peroxidase reductase (ET rxns) > Inserted into polypeptide during translation but not specified by codon 22nd AA: L-Pyrollysine - genetic code for pyrrolysine: in Archae microbes named Methanosarcina barkeri which produce methane or natural gas

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a functional group that contains a C-N double bond with the N atom connected to an aryl or alkyl group— but not H  A mechanism used by enzymes to catalyze rxn occurs through the formation of imines (Schiff bases)

-basic polar: side chain contains an amino group Serine Threonine Asparagine Glutamine Cysteine Tyrosine

Reversible oxidation of 2 cysteine side chain thiols to form cysteine

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GRP 1 (nonpolar side chains)  Aliphatic HC side  Alanine  Valine  Leucine  Isoleucine  Proline aliphatic cyclic structure, N bonded to 2 C (secondary amine)  Tryptophan : indole ring in R  Pheneylalanine (aromatic HC With benzene ring)  Methionine R contains S (aliphatic )  Glycine (no polar side chain) GRP 2 (Polar      

R are neutral) Serine (OH) Threonine (OH) Asparagine (amide) Glutamine (amide) Cysteine (SH) Tyrosine (OH is phenol)

GRP 3 (acidic Carboxyl in R)  Glutamate  aspartate GRP 4 (Basic side chains)  Histidine (imidazole)  Lysine  Arginine (guanidino) Classification: 1. Whether the R group is acidic, basic, neutral-polar, or neutral-nonpolar -AA with nonpolar R are classified as hydrophobic -those with polar side chains are classified as hydrophilic 2. According to Timberlake -acidic polar: side chain contains a carboxylic acid

Non-polar -R group is made up of only carbon and hydrogen -non polar since there is very little polarity associated with carbon-carbon and carbonhydrogen bonds (therefore hydrophobic) -Phe, Met, Ile, Leu, Val are very hydrophobic Isoleucine Phenylalanine Methionine Leucine Valine Glycine Alanine Proline Tryptophan

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Heteroatoms -Sulfur -Nitrogen >Contribute very little polarity to the side chain Heteroatoms in Methionine (sulfur) and tryptophan (Nitrogen) -overall behavior of these AA: nonpolar Side chains which contain more polar functional groups such as acids, amine, alcohol, and thiol provide locations for a polar water molecule to hydrogen bond They are thus somewhat hydrophilic (like the OH groups in a sugar) These side chains are important in making a protein sufficiently water soluble to operate effectively inside a cell

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Acidic Polar -Aspartic Acid -Glutamic Acid  acidic AA have side chains that contain a carboxyl group in addition to the one next to the amino group  at cellular pH is near neutral (ph-7) the carboxyl group is dissociated so that the R group has a negative charge (present as the carboxylate anion)  secondary carboxylic group is weaker acid  carboxylate side chain is important in interaction with metal in many enzymes, in ionic interactions Basic Polar -Lysine -Arginine -Histidine  side chain include an amino group. These amino groups are also ionized (present as the ammonium ion) at neutral pH  Basic AA are positively charged as a result of the dissociation of the amino group in their side chain  The ionized groups are quite polar.  They make side chain quite hydrophilic.  Acid and base chains are ionic and therefore hydrophilic.

 diamino, monocarboxylic: L-lys, L-arg, L-his           

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Lysine: ammonium ion Arginine: guanidium group Histidine: imidazolium group

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Cystine formed by mild oxidation of 2 sulfihydryl group forming disulfide bond -hair perm creates disulfide linkages

Structure (Devlin)  Monoamino, monocarboxylic –Gly, L – Ala  Unsubstituted: L-val, lue, ile  Heterocyclic: -Pro, -Phe  Aromatic: Tyr, Trp  Thiother: Met  Hydroxy: ser, thr  Mercapto: cys  Carboxamide: asn, L-gln  monoamino, dicarboxylic: L-asp, L-glu

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All 3 basic AA (KRH) have a positive charge on the Nitrogen in the R side chain Serine is formed by adding a hydroxyl group to Alanine Cysteine is formed by replacing the O with S in Serine Threonine is formed by adding CH3 to Serine Valine has a V shaped side chain Leucine has a Y shaped side chain Isoleucine has an upside down Lshaped side chain Proline is shaped like a pentagon with the amino group incorporated in the ring cyclic amino acid. It is nonpolar (wt aliphatic grp) one of the ambivalent amino acids, meaning that it can be inside or outside of a protein molecule occurs in proteins frequently in turns or bends, which are often on the surface Gly: sharp bends, allow more flexibility When we replace the H with a methyl group, we get Alanine we add a phenyl group to alanine, we get phenylalanine. add a hydroxyl group to Phe, we get Tyrosine Aspartic acid is formed by adding a carboxyl ion to Alanine: Glutamic acid is formed by inserting another CH2 into Aspartic acid:

Aromatic • nonpolar • To different degrees, all aromatic amino acids absorb ultraviolet light. Tyrosine and tryptophan absorb more than do phenylalanine • tryptophan is responsible for most of the absorbance of ultraviolet light ( 280 nm) by proteins • Tyrosine is the only one of the aromatic amino acids with an ionizable side chain. Tyrosine is

one of three hydroxyl containing amino acids hydroxyl groups – serine and threonine are so high that they are generally regarded as nonionizing Structure of Side Chain (Koolman) 1. Aliphatic (do not contain N, O, S in side chain) – branched-chain AA or BCAA sometimes used to refer to AA having aliphatic side chains that are nonlinear -Gly, -Ala, -Val, -Leu, -Ile 2. Sulfur containing -Cys, -Met 3. Aromatic (benzene ring in side chain) -Phe, -Tyr, -Trp 4. Neutral (hydroxyl or amide groups in side chain) -Ser, -Thr, -Asn, -Gln 5. Acidic (carboxylate groups in side chain) -Asp, -Glu 6. Basic -Lys, -Arg 7. Imino acid -Pro •

polar and positively charged at pH values below their pKa's, and are very hydrophilic

Lysine:  the side chain of lysine has a marked hydrocarbon character, so it is often found NEAR the surface, with the amino group of the side chain in contact with solvent Histidine,  essential aa, has as a positively charged imidazole functional group.  unprotonated imidazole is nucleophilic and can serve as a general base  protonated form can serve as a general acid  serve a role in stabilizing the folded structures of proteins

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Cyclic AA -Imino acid -the only proteinogenic AA whose side group links to the α-amino group and thus the only containing secondary amine at the position

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Most plant proteins have insufficient amounts of lysine and tryptophan; thus strict vegetarians should ensure that their diet contains sufficient amounts of these 2 AA Isoelectric AA fully ionized and internally neutralized by their own amino and carboxyl groups

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 depending on the number of the protons they can give up, we define monoprotic, diprotic and triprotic acids  monoprotic, (e.g. acetic acid or ammonium) have only one dissociable group  diprotic (carbonic acid, bicarbonate, glycine) have two dissociable groups and  triprotic (e.g. phosphoric acid) have three dissociable groups. • In the case of multiple pK values they are designated by indices: pK1, pK2, pK3, etc • For amino acids, the pK1 constant refers to its carboxyl (-COOH) group • pK2 refers to its amino (-NH3) group • the pK3 is the pK value of its side chain • Both the a-amino group and the acarboxyl group are ionizable • a-COOH group: a weak acid, can DONATE its proton, with a pKa of about 2-3 • a-NH2 group: a weak base (there is an unshared pair of electrons on the N; the neutral amino group) can ACCEPT a proton • side chain ionizable groups: only 7 amino acids  Asp, glu  His, lys, arg  Cys, tyr

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Glycine (Gly, G) the simplest and smallest of all AA and the only one which is not optically active since it has a single H atom on its side chain

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Alanine (Ala; A) has a methyl group as its side chain.

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Valine (Val; V) has a slightly longer side chain again and this time there is a branch. AS the aliphatic side chains get longer they are also more hydrophobic. Leucine (Leu; L) is very similar to Valine except it has another methyl group attached to the side chain

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Aromatic AA, aromatic ring as part of their side chains, are highly hydrophobic.  Phenylalanine (Phe; F) first of all the aromatic AA -contains a phenyl ring attached to a methylene group  Tyrosine (Tyr, Y) contains a hydroxyl group at the end of the phenyl ring (makes tyrosine less hydrophobic) -is a reactive group (the side chains so far have all been unreactive) Sulfur containing  Cysteine (Cys; C) sulphydryl group (-SH) is extremely reactive, can form hydrogen bonds and disulphide bridges -even though the –SH group can form H bonds the long aliphatic part of the side chain makes it quite hydrophobic 

Methionine (Met; M) is a very special amino acid -the “start” AA in the process of translation therefore begins every single protein -Sulfur atom in a thioether linkage an is relatively unreactive -has a high hydrophobic side chain

Hydrophilic AA, neutral, those which are acidic and those which are basic Basic AA contain side chains which are positively charged at physiological pH -the pKa of Histidine which is between 6 and 7 in proteins means that it is able to

accept or donate proteins at physiological pH. For this reason histidine is often found at the active site of enzymes.  Lysine (Lys; K) has one of the longest side chain -although side chain appears to be a hydrophobic hydrocarbon chain it is very polar because of the terminal amino group  Histidine (His; H) imidazole ring which often sits inside the active site of an enzyme (helps bond to be broken or made) -the pKa of Histidine is between 6 and 7 in proteins (is able to accept or donate proteins at physiological pH) -It can do this because it can exist in 2 states, uncharged or positively charged 

Arginine (Arg; R) has largest of all side chains -guanidino group attached to the side chain -has a high pKa value and is positively charged at physiological pH

Neutral Polar AA,not charged at physiological pH however they all have groups in their side chains which are polar and can form H bonds so are classed as hydrophilic 

Serine (Ser; S) contains an aliphatic chain with a hydroxyl group -the OH group makes the AA highly reactive and hydrophilic as it readily forms H bonds

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Threonine (Thr; T) neutral AA which has a highly reactive (and highly hydrophilic) hydroxyl group -contains 2 centers of asymmetry (2 asymmetric C atoms shared only by isoleucine)

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Asparagine (Asn; N) is the amide derivative of Aspartic acid, side chain is amidated -resulting amide is uncharged -there is a terminal amide group as opposed to the carboxyl group on aspartate

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Glutamine (Gln; Q) is similar to Asparagine with a terminal amide (instead of a carboxyl group as in glutamate) -These 2 are called the amide derivatives of their parent AA

Aromatic AA (Trp, Tyr, Phe) absorb light in the near UV region of the spectrum (250-300 mm)  Trp has highest molar absorptivity followed by Tyr, with Phe making only a small contribution  Disulfide bonds (between Cys residues in proteins) also absorb in the UV range, but much less than the aromatics Posttranslational modifications of AA side chains  Chemical modifications AFTER biosynthesis of proteins  Occur a few AA residues in some proteins AA  20 (biologically essential)  Can synthesize 12 -amphibolic intermediates of glycolysis and CAC (9) -3 (cysteine, tyrosine, hydrolysine) from nutrionally essential  AA that we produce: 1. Alanine 2. Asparagine 3. Aspartic acid 4. Cysteine (from methionine) 5. Glutamic acid 6. Glutamine 7. Glycine 8. Proline 9. Serine 10. Tyrosine (Tyrosine is produced from phenylalanine, so if the diet is deficient in phenylalanine, tyrosine will be required) 11. Arginine 12. Formed posttranslational processing of collagen -Hydroxylysine -hydroxytyrosine

Essential AA 1. Histidine 2. Isoleucine 3. Leucine 4. Lysine 5. Methionine 6. Phenylalanine 7. Threonine 8. Tryptophan 9. Valine 10. Arginine (required for the young but not for adult) Isoelectric point (pI) • pI = the pH exactly halfway between the two pKa values surrounding the zero net charge equivalence point on the titration curve • If pH < pI, net charge is positive (more + than - charges) • If pH > pI, net charge is negative (more - than + charges)

pI = pK1 + pK2/ 2 = 2.34 + 9.60/2 = 5.97 At pH < pK1 COOH – NH3 + +1 At pH betw pK1 and pI half of COOH and half COO and NH3 (0.5- and 1+) At pH betw pK1 and pK2 = COO, NH3+ 0 Above pK2 COO- and NH2 -1

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• pH betw pK1 and pK2 COO, COOH, NH3 : 0 • pH >pK2 COO, COO,NH3 : -1 • pH above pK3 COO, COO, NH2 : -2

the remaining H+ attaches itself to the weak base, giving weak acids as one of the products Ex. Ammonia, NH3+ (most of the ammonia in solution remains unionized and a small fraction ionizes to form NH4+ and OH- ions -when an acid reacts with a base produces a salt and water, it is called neutralization

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• Aspartic acid • COOH,NH3,COOH • At pHpK2 COO, COO, NH3: -1 • pH above pK3 COO, COO, NH2: -2 • pK1 2.1 (+1 and 0) • pK2 3.9 (0 and -1) • pK3 9.8 (-1 and -2) pI = pK1 + pK2/2 = 2.1 + 3.9 = 3

– Dipolar form in which amino and carboxyl are ionzed – Net charge = 0 – The pH at which an AA is electrically neutral : the sum of the + charges = the sum of the neg charge – Region of buffereing : pKa +/- 1

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Strong acids ionize completely in water Ex. Hydrochloric acid (HCl)

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Weak acids ionize partially in water Ex. Acetic acid (most of the acetic acid in solution remains unionized and a small fraction ionizes to form CH3COO- and H3O+ ions

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pKa1 of Glutamic Acid = 2.2 (COOH) pKa2 of Glutamic Acid = 4.3 (COOH) pKa3 of Glutamic Acid = 9.7 (NH2) pI=pK1 +pK2 = 3.2 • At pH
Acid+Base  Salt+water  

ACIDS AND BASES  Arrhenius: -that acids are compounds that contain H and can dissolve in water to relase H into solution -bases releases hydroxide ions (OH) in solutuion  Bronsted-Lowry: -Base: a molecule or ion that ACCEPTS H ions from solution -Acid: substances that can donate a H ion  Lewis Model -acid: accepts a pair of electrons -base: donates a pair of electrons

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Strong bases ionize completely in water. Ex. Sodium hydroxide, NaOH (contains entirely Na and OH ions) Weak Bases ionize partially in water -do not furnish OH-ions by dissociation. They react with water to furnish the OH ions -when a weak base reacts with water the OH- comes from the water and

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pH and pOH are quantitative ways to express solutions of acids and bases Under the Bronsted-Lowry definition both acids and bases are related to the concentration of H ions present Acids increase the concentrations of H ions Bases decrease the concentration of H ions (by accepting them The acidity and basicity of something therefore can be measured by its H ion concentration Concentration of H ion: -measure of active acidity or basicity -# of moles of H ion/liter of solution -expressed as pH (the neg log to the base 10 of H ion concentration) pH scale is a more convenient way than using exponential notation for expressing concentration of H ions -Danish biochemist Soren Sorensen invented the pH scale for measuring acifity. The pH scale is described by the formula: pH=-log[H+] -H ion concentration in units of moles of H+ per liter of solution For any Hydrogen ion concentration of - 1 x 10-n=[H+] - the pH is equal to pH=n -pH scale The pH scale ranges from 0 to 14 pH and [H+] are inversely relatedlower means higher [H+] therefore: -substances with a pH between 0 and less than 7: ACIDIC -substances with a pH greater than 7 and up to 14 are BASES (higher pH means lower [H+]

-right in the middle at pH 7 are NEUTRAL substances, ex. Pure water -NEUTRAL: H=OH (at 7pH under T 25 c) pH affects functions, interactions -change in pH may change interactions -dipole-dipole interactions might change to ionic -if pH of solution matches pKa (of side chain) half is protonated and half is deprotonated, -at lower pH, more than half is protonated -at higher pH more than half is deprotonated Titration: methodof (usually) finding the concentration of an unknown liquid by comparing it with a known liquid

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Acid dissociation constant, Ka, (acidity constant, or acidionization constant) -is a quantitative measure of the strength of an acid in solution -amount of H released -specific type of equilibrium constant that measures the propensity of something larger to separate (dissociate) reversibly into smaller components Ex. HA  A- + H+ -Ha represent acetic acid, and A- the acetate ion -the chemical species HA, A- and H+ are said to be in equilibrium when their concentration do not change with the passing of time Equilibrium constant for the acid-base equilibrium of an acid with its conjugate base Quotient of the qequilibrium concentrations, denoted by [HA],[A-] and [H+] [𝐻 +][A−] [𝐻𝐴] When protonated and conjugated are equal the conc of H is= to Ka Conjugate base=undissociated COO- = COOH Ka =

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K = H+ (conc.) -logK = - log H pK = pH Prevailing H is numerically = to K Experimental determination of pKa values is commonly performed by means of titrations -calculate pH of weak acid after addition of strong base (HendersonHasselbach eq) -quantitative relationship between pH and conc weak acid and its base [𝐴𝐻] [𝐴] Isoelectric AA fully ionized and internally neutralized by their own amino and carboxyl groups Zwitterion: overall change is 0 Both the a-amino group (amino group substituent on the aC) and the acarboxyl group (carboxyl substituent on the aC) are ionizable A-COOH group: a weak acid, can DONATE its proton with a PKA of about 2-3 A-NH2 group: weak base (there’s an unshared pair of electrons on the N: the neutral amino group can ACCEPT a proton) Isoelectric point- pH at which the predominant from zwitterion pH = pKa − log

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PROTEINS • linear polymers built from 20 different L-α-amino acids Peptide bond • Primary linkage of all protein structures • chemical bond formed between the carboxyl groups and amino groups of neighboring amino acids • dehydration synthesis reaction (condensation reaction) • The resulting CO-NH bond is called a peptide bond, and the resulting molecule is an amide (residue)  The four-atom functional group C(=O)NH- is called an amide group or (in the context of proteins) a peptide group  These bonds are rigid and planar, due to electron sharing between the carboxyl carbon and the amide nitrogens which contribute to the bond and give it a partial double-bond character  Peptide bond is planar/flat: due to its partial double-bond  The electrons from the double bond to Oxygen migrate to the bond between th C and the N producing partial + (N) and -ve (O) charges there is not free rotation about the C -N bond  the group of atoms about the peptide bond can exist in the cis or trans configurations  The most stable conformation is planar and trans  Proline is mostly in cis  Isomerization :about the C-N bond  Left : cis  Right: trans

• chain torsion angles of a polypeptide • These are phi, psi, and omega (W) • Phi and psi (N-Ca and Ca-C bonds) relatively are free to rotate • Free rotation:  Alpha C –CO: Psi angle  Alpha C – N: phi angle  Rachamandran angles • Free rotation can occur about the bonds that connect the -C with the -N and with the -carbonyl C • polypeptide chain is thus a semirigid structure with two-thirds of the atoms of the backbone held in a fixed planar relationship • Each peptide chain has one amino terminus and one carboxyl terminus. • These are the only free alpha amino and alpha carboxyl groups in the peptide • All of the others are involved in the peptide bonds between amino acids • peptide is a compound consisting of 2 or more amino acids • Oligopeptides have 10 or more amino acids (20) • Polypeptides are chains of about more 20 to 50 • proteins are peptides consisting of more than 100 amino acids Classification accdg to composition • Simple  Consist solely of AA  Albumin  Globulin  Glutelin (glutenin-wheat, oryzeninrice  Protamine  Albuminoids, scleroprotiens: keratin, elastin, collagen  Histones, globin

• Conjugated – With non protein prosthetic grp • Nucleoprotein (nucleic acid with protamine/histones) in chromatin • Glycoprotein/mucoprotein 9ground substance of connective tissues) • Phosphoproteins ( H3PO4) • Chromoprotein( with hematin) – Hemoglobin, cytochromes, rhodopsin • Lipoproteins – With Fats, phospholipids • Metalloproteins – Ceruloplasmin, siderophilin • Derived – Primary derivatives • Intramolecular rearrangement thru hydrolysis • Denatured protein – Proteans  From action of water, dilute acids or enzymes  Myosan, edestan – Metaproteans  From further nydrolysis  Acid albuminates/alkali – Coagulated  From action of heat, alcohol, uv, shaking  Cooked egg albumin, meat – Secondary • From more extensive hydrolysis, fragments of original ptotein which are soluble in water, non coagulable by heat – Primary proteoses – Peptones – peptides Classification accdg to function 1. Structural proteins – Supporting filaments • Internal network : cytoskeleton, cellular shape and physical integrity • Actin and myosin : contractile machinery of muscles – examples • Collagen • elastin • Alpha keratin (hair, nail, feather) • Fibroin (silk of spider web) • Glycoprotein (cell coat) • mucoprotein

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2. Transport • Serum albumin • Hemoglobin • Lipoprotein • Ceruloplasmin • Iron binding globulin 3. enzymes - >2000 4. Nutrient and storage • Ovalbumin • Ferritin • Gliadin • zein 5. Contractile or motile – Actin and myosin 6. Protective – Ig, fibrinogen, thrombin 7. Hormones 8. Toxins 9. Receptors Classification accdg to shape 1. globular – Chains are tightly folded spherical molecules – Most have motile function – Soluble in aqueous solution – Nearly all are enzymes – Antibodies – Nutrient storage – Hgb 2. Fibrous – Chains are arranged in parallel along a single axis to form long fibers or sheets • Physically tough • Insoluble in water – Basic structural elements in connective tissues • Collagen of tendon and bone matrix • Alpha keratin of hair, skin, nails and feathers • Elastin of elastic tissues 3. those which fall between fibrous and globular – Long rodlike structures: myosin, fibrinogen Physical properties – Tasteless, bitter (hydrolysates) – Colorless, amorphous (some colored and crystalline) – Insoluble in fat solvents – Varied solubility in water – amphoteric

solubility • Depends on the AA components on the surface – High hydrophobic on the surface : low solubility – Charged, polar surface residues increase solubility Precipitation: “salting out” • Addition of neutral salt (NH4SO4) – Increased reaction of charges of protein with the salt and exposes hydrophobic areas on prtein surface which aggregate and precipitate Coagulation point – Characteristic of individual proteins • Acids lower • Alkali raises • Hydration lowers Color reactions – Biuret : Protein in serum forms a violet colored complex with cupric ions in an alkaline solution. The intensity is proportional to the amount of protein present Ampholytes – In acid solution acts as base – In alkaline acts as acid Biological Roles of proteins • Acts as Catalysts. • Fibrous proteins serve as components of the tissues holding the skeletal elements together • The nucleoproteins serve as carriers of genetic characters and govern inheritance. • Performs transport function via catalytic activity or as adsorbent. • Acts as hormones and regulate the growth of plants and animals besides controlling many other physiological functions. • Under condition of non-digestion the protein accumulate inside cells and produce toxicity (e.g.Venoms of snakes and insects). Bonds in the structure • Strong bonds 1. peptide 2. Disulfide

• Interconnect 2 parallel chains thru cysteine residues within each polypeptide • Relatively stable Weak bonds – 1. Hydrogen bonds • Sharing of bonds between the Nitrogen and carbonyl Oxygen of the same or different chains • They are significant due to extremely large number of H bonds • Broken during denaturation – Hydrophobic bonds • Nonpolar side chains of neutral amino acids are associated with one another • Maintain protein structure – Electrostatic bonds • Salt bonds formed between oppositely charged groups in the side chain of amino acids • Broken by denaturation Folding • These linear chain of amino acids interact with each other and their surroundings in the cell to produce a well-defined, three dimensional shape • The shape into which a protein naturally folds is known as its native state • The three-dimensional structure is determined by the sequence of the amino acids. • mechanism not understood • the mechanism depends equally on  the characteristics of the cytosol, the nature of the primary solvent (water or lipid),  macromolecular crowding  the concentration of salts  temperature  molecular chaperones assist in the folding of proteins • Most folded proteins have a hydrophobic core in which side chain packing stabilizes the folded state • the N-terminus of the protein begins to fold while the C-terminal portion of the protein is still being synthesized by the ribosome

Levels of Orders

Primary structure:

• the amino acid sequence • Aminoacyl residues: replace the suffixes with –yl • Peptides are named as derivatives of the carboxyl terminal aminoacyl residue – Lysyl-leucyl-glutamine • most fundamental, all proteins have • Amino acids that are ionizable in protein or polypeptide:  In side chain (7) Basic: Histidine, arginine, lysine Acidic: Aspartate, glutamate Thiol: Cysteine tyrosine  Terminal amino acids

Secondary structure:

• Due to formation of H bonds between one peptide bond with another within chain • carbonyl group (O) of amino-acid makes a hydrogen bond with the amino group (H) of another amino-acid • Dictated by the primary structure • The most common examples are the  alpha helix and  beta sheet • Alpha helix  R-handed coiled or spiral conformation (helix), in which every backbone N-H group donates a H bond to the backbone C=O group of the aa 4 residues apart  formed due to H bonds formed between O of the CO and the H atom of the peptide bond N of the 4th residue down the chain  backbone chain i is a R-handed helical conformation  Each turn of the helix comprises 3.6 amino acids  stiff, rod-like structures

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• Proline induces bends in alpha helices • Since peptide bond N of proline lacks a H atom to contribute to H bond, proline can only be accomodated within first turn of alpha helix • When present somewhere else it disrupts the conformation of the helix, producing a bend • Other AA that may disrupt regular pattern of helix  Group of isoleucine causes steric hindraance due to bulky side chains  Glycine due to small R which allows movement destabilize  Group of acidic AA destabilize due to negative charges which repel each other  Lysine, Arg. Serine, threonine • Many alpha helices have predominantly hydrophobic R on one side and predominantly hydrophilic on the other therefore amphipathic  Well adapted to formation of interfaces between polar and nonpolar regions - Hydrophobic interior of a protein and its aqueous environment - Clusters can create channel, pore that allow polar molecules to pass thru hydrophobic cell membranes • Other helices - the 310 helix and n helix - ends of α helices due to unfavorable backbone packing in the center of the helix beta strand • (also β strand) is a stretch of polypeptide chain typically 3 to 10 aminoacids long with backbone in an almost fully extended conformation • β sheet refers to an assembly of at least two such β strands that are hydrogenbonded (or H-bonded) to each other - between carbonyl O and amide H of peptide bonds formed with adjacent segments • Most are not perfectly flat but with right-handed twist • Clusters of twisted strands form the core of many globular proteins • in structure of the enzyme triose phosphate isomerase

• loops and turns are required to connect alpha-helices and beta-strands • These are usually found on the surface of the protein • Therefore they contain mainly hydrophilic residues and are often binding sites Turns and bends: • Short segments of AA that join two units of secondary structure (2 adjacent strands of an antiparallel sheets) • A turn reverses the direction of a helix/strand  is stabilised by a hydrogen bond between the backbone CO of one amino acid with the NH of the next  Beta turn: involves 4 residues in which the 1st is H-bonded to the 4th (tight 180-degree turn). Proline and glycine • A loop is a much larger sequence which changes the direction of the helix/strand  There is no regular structure or sequence of amino acids in these regions  connect adjacent regions of secondary structure  Irregular conformation • Tight turns and loose, flexible loops link the more "regular" secondary structure elements • Proline and glycine are known as "helix breakers" because they disrupt the regularity of the α helical backbone conformation - have unusual conformational abilities - commonly found in turns • Amino acids that prefer to adopt helical conformations in proteins : methionine, alanine, leucine, glutamate and lysine • prefer to adopt β-strand conformations - the large aromatic residues (tryptophan, tyrosine and phenylalanine) - Cβ-branched amino acids (isoleucine, valine, and threonine) supersecondary structure • formed by the combi of several neighbouring alpha-helices/ beta-sheets into a particular geometric arrangement

• arrangements of 2 or 3 secondary structures present in protein structures • term "motif" is often used to describe super-secondary structures • connections between  long coiled-coil regions  alpha-helices connecting the beta-sheets

• refers to the overall three-dimensional structure of a single polypeptide chain. Regions of regular secondary structure (e.g. alpha helicies and beta sheets) "

Tertiary structure

• fold up" along with the "randomly" coiled regions into a compact, generally globular structure. • Tertiary structure is the "global" folding of a single polypeptide chain - to assemble the diff secondary struc elements in a particular arrangement - the overall shape of a single protein molecule; often used as synonymous with the term fold - the spatial relationship of the secondary structures to one another • domain is the unit of tertiary structure • Bonds that stabilize tertiary level  Hydrophobic  Ionic  H bonds  disulffide • controls the basic function of the protein

Quaternary structure

• involves the association of two or more polypeptide chains (tertiary structure) into a (multi-subunit) active structure • Not all proteins exhibit quaternary structure • Stabilized by;  D bonding, van der Walls interactions  ionic bonding (between charged Rs)  Hydrophobic (between non-polar Rs)  disulfide bonds between cysteine residues in different polypeptide chains

• units of tertiary structure aggregate to form homo- or hetero- multimers • Hetero-multimers  different tertiary domains aggregating together to form a unit • Homo-multimers  more common to find copies of the same tertiary domain associating non-covalently Separating proteins • Size - Ultrafiltration - centrifugation - Size exclusion chromatography  through a chromatography column filled with porous beads  Diff. Molecules enter the pores according to how easily they can enter  Larger pass thru column  Diff in the time required to pass or the condition required to elute the protein from the column • charge - solubility, ion exchange chromatography, and electrophoresis - Proteins are least soluble at their isoelectric point. At the isoelectric point, many proteins precipitate from solution - Net charge (abov and below Pi)  ion exchange chromatography and electrophoresis - In ion exchange chromatography, the greater the magnitude of the charge, the slower a protein moves through a column - Electrophoresis  In electrostatic field  Molecules with no net charge do not move  with a net positive charge move toward the negative end  those with a net negative charge move toward the positive end  magnitude of the net charge determines how fast the species moves

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• Sequence of AA - Denaturing the protein • cleaving the disulfide linkages (oxidation of the S to SO3 • Detrmine N terminal (Edman degradation. tags the N-terminal residue then cleaved) -use Sanger’s reagent, dansyl chloride, and leucine aminopeptidase • C terminal - Akabori reaction (hydrazinolysis) and reduction with lithium aluminum hydride tag - selectively cleave the C-terminal residue using the enzyme carboxypeptidase • cleave the polypeptide into smaller fragments and determine the amino acid composition and sequence of each fragment • Partial acid hydrolysis randomly cleaves the protein chain into a number of fragments • Trypsin, a digestive enzyme, specifically cleaves on the C-side of arginine or lysine • chymotrypsin preferentially cleaves residues containing aromatic rings (tyrosine, phenylalanine, and tryptophan). It slowly cleaves other residues especially leucine. Clostripain cleaves positively charged amino acids, especially arginine; It cleaves lysine more slowly • Chemical :  cyanogen bromide, hydroxylamine, and heating an acidic solution  Cyanogen bromide specifically attacks methionine • Apply Edman degradation to the fragments Collagen • Main protein in CT of animals • Most abundant protein in mammals (2535%) • Tropocollagen: fundamental unit of collagen (3 chains) • Alpha helix : L-handed helix • 3 entwine to form R-handed triple helix -Resists unwinding: it and its 3 chains are coiled in opposite directions

• located in the extracellular matrix of CT. • Most abundant protein in body • classification of an extracellular matrix protein as a collagen is based on the presence a distinctive triple-helical conformation • The collagen triple helix consists of three polypeptide chains super coiled about a common axis and linked by hydrogen bonds. • 28 types • 90% are type I-IV • Specific types are associated with particular tissues • The most prevalent and well-studied collagens belong to the fibril-forming or interstitial collagen family Type I • is the most common fibril-forming collagen (90%) • Its fibrils make up the mineralized matrix in bone, the strong parallel bundles of fibers in tendon, and the plywood-like alternating layers in the transparent cornea  Dentin  fascia  Scar  skin Type II • is the major fibril-forming collagen in cartilage • Vitreous body, nucleus pulposus Type III • reticulin • is found in blood vessels and skin, together with type I, granulation tissue Type IV • Basement membranes, which serve to separate cell layers and act as filtration barriers - organized into a network or mesh-like sheet structure - In the kidney • Every 3rd residue is glycine ( gly-X-pro) or Gly-X-Hyp) (X hydroxyproline or hydroxylysine) • 1/3 is glycine • Proline 9% of collagen • OH of Hyp : in forming H bonds • OH of Hyl attachment sites for polysaccharides

Synthesis • In fibroblasts  Translation of a-chains (preprocollagen) (RER)  Hydroxylation (ER) rqrs vit C  Glycosylation (ER)  Exocytosis into extracellular • Outside fibroblast  Cleavage of terminal of procollagen to tropocollagen (insoluble) • Tropocollagen chains associate to form microfibrils  Cross linking covalent lysinehydroxylysine linkage Disorders Ehlers Danlos syndrome - Faulty collagen synthesis  Hyperextensible skin  Easy bruising  Hypermobile joints • Autosomial dominant or recessive • Type III most frequently affected

hemoglobin • Metalloprotein (iron) • oxygen-transport in the red blood cells of vertebrates • In mammals, the protein makes up about 97% of the red blood cell’s dry content, and around 35% of the total content (including water) • four globular protein subunits • four polypeptide chains: two alpha chains, each with 141 amino acids and two beta chains, each with 146 amino acids. • Each subunit is composed of a protein chain tightly associated with a nonprotein heme group Keratin • particularly abundant in the proteins of hair, hooves, and the keratin of the skin

Osteogenesis imperfecta • Brittle bone disease • Autosomal dominant , most common form • Abnormal type I ; (type II :fatal utero)  Multiple fractures (during birth)  Blue sclerae (translucent connective tissue over choroid)  Hearing loss (abn middle ear bones)  Lack dentin – dental abn Alport’s sundrome • Abnormal type IV • (basement membrane of kidney, ears, eyes) • Most common form X-linked recessive • Nephritis, deafness, ocular Vit C deficiency • Prolyl and lysyl hydroxylases: poor function, therefore no cross links Myoglobin • a single-chain globular protein of 153 aa, containing a heme (Fe-containing porphyrin) prosthetic grp in the center around wc remaining apoprotein folds • 8 alpha helices and a hydrophobic core • primary oxygen-carrying pigment of muscle tissues.

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