Amino Acids and Proteins

 

 AMINO ACIDS  ACIDS  PROTEINS  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 from same    All are constructed from 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

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

>solubility >solubili ty in water -the identity/distinctive identity/distinctive >size, structure   helps to dictate the folding of the protein

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 Alpha Carbon Carbon atom  atom     A hydrogen atom    Amino group (hence (hence “amino” acid acid))  -the amino group (-NH 2) accepts a proton and becomes (-NH3+)   Carboxyl group (-COOH) -this gives up a proton and is thus an acid (hence amino “acid”)  “acid”)   -becomes dissociated (-COO-)   R group 

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Threonine  and Isoleucine Threonine and Isoleucine have  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  aminos  D form proteins are proteins are produced by exotic sea-dwelling organisms such as cone snails -abundant components of the peptidoglycan cell walls of bacteria -D-serine D-serine may  may act as a neurotransmitter neurotransmi tter in the brain

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 AMINO ACIDS  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 chain R  H

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No asymmetric carbon: GLYCINE is not asymmetric  Alpha carbon is asymmetric 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 superimposab le mirror images) Optically active 2 different arrangements of the groups around the alpha carbon: 1.  Enantiomers Enantiomers-mirror -mirror image 2.  Stereoisomers Non-superimposable

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D-AA that occur naturally -Free: D serine, D asp (brain tissue) -D ala, D glu (cell w all 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) (imidazolium) can serve as a general acid acid form, the imidazolium ion (B), is a resonance hybrid imidazole Conjugate Conjugate base (A or C) Either of the two ring nitrogens can release a proton (H+) to produce the conjugate base form

   A-amino acids because because they have a primary amino group (-NH 2) and a carboxylic acid group (-COOH) attached to the alpha carbon    Amino acid common 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)

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group (-R)    Variable side group   determines: -its special properties -size, shape, reactivity >electric charge

 

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 AA are L or D depending depending on the position of the amino group Regular tetrahedron. 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  “handedness” is 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

Since the amino group in proline is involved in two carbon-nitrogen 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 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  Acid –  – base  base -Amino and COOH groups -pKa   pKa -  COOH : ~2.2 -   Amino: ~9.4 At pH 7 the amino is protonated protonated   At NH3+ and carboxyl is in COO(conjugate base or carboxylate form) AA with single single amino and single single   AA carboxyl group will have net charge of zero : Zwitterion; which are electrically neutral   In an electric field : stationary

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

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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 2.2: COO-  predominates  At pH between 2.2-9.4 the predominant state of the molecule: COO- and NH3+  -zwitterions zwitterions   Can be in 3 general forms: 1.  Protonated form (dipolar with charge): -they can dissociate -NH3+ and COO-  2.  Unprotonated/ Neutral form: -they cannot dissociate -NH2 and COOH 3.   Amino group: group: ~9.6 pH>NH3+  Carboxyl group: __________ 

 At low pH the amino and carboxyl carboxyl groups will be protonated and the molecules will be in the acid form COOH: ~2.2  As the pH is increased (beyond (beyond 2.2, COOH dissociates) towards neutrality, the AA become zwitterions having both negative and positive charges  AS the pH increase further further (beyond pK of aa) NH3 dissociates 20 proteinogenic AA 21st amino acid Selenocysteine -Found in some proteins > Peroxidase reductase (ET rxns) > Inserted into polypeptide polypeptide during translation but not specified by co don 22nd AA: L-Pyrollysine L-Pyrollysine   - genetic code for pyrrolysine: in  Archae microbes named named  which Methanosarcina barkeri  which produce methane or natural gas

Reactions:   Reactions:    Between AA and carboxyl groups -formation of peptide bonds   Peptide bond  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 atoms are nearly planar and there is no rotation about the peptide bond

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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   A catalyze rxn occurs through the formation of imines (Schiff ( 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

a. 

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

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Isoleucine Phenylalanine Methionine Leucine  Valine Glycine  Alanine Proline Tryptophan

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GRP 2 (Polar (Polar R are neutral)  neutral)  Serine (OH)   Threonine (OH)   (amide)    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   (guanidino)    Arginine (guanidino)

 

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Non-polar   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 Val are very hydrophobic

Classification: 1.  Whether the R group is acidic, basic, neutral-polar,, or neutral-nonpolar neutral-polar neutral-nonpolar -AA with nonpolar R are classified as hydrophobic -those with polar side chains are classified as hydrophilic 2.   According to Timberlake Timberlake -acidic polar: side chain contains a carboxylic acid

 

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Heteroatoms -Sulfur -Nitrogen >Contribute very little polarity to the side chain  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  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

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

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Basic Polar  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 chains are ionic and therefore hydrophilic. 

 

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

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one of three hydroxyl containing amino acids

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 All 3 basic AA (KRH) (KRH) have a positive charge on the Nitrogen in the R side chain Serine is Serine  is formed by adding a hydroxyl group to Alanine Cysteine is Cysteine  is formed by replacing the O with S in Serine Threonine is formed by adding CH3  to Serine  Valine has  Valine  has a V shaped side chain Leucine has 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 phenylalanin phenylalanine. e. add a hydroxyl group to Phe, we get Tyrosine  Aspartic acid is formed 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 Tyrosine  and tryptophan absorb more than do phenylalanine •  tryptophan tryptophan is  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. chain. Tyrosine is

 

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

 

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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hydroxyl groups  –   serine and threonine are so high that they are generally regarded as nonionizing 

Structure of Side Chain (Koolman) 1.   Aliphatic  Aliphatic (do  (do not contain N, O, S in side chain)  –  – branched-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  Aromatic (benzene  (benzene ring in side chain) -Phe, -Tyr, -Trp 4.  Neutral (hydroxyl or amide groups in side chain) -Ser, -Thr, -Asn, -Gln 5.   Acidic  Acidic (carboxylate  (carboxylate groups in side chain) - Asp, -Glu 6.  Basic Basic   -Lys, -Arg 7.  Imino acid  acid  -Pro

 

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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 nucleophilicc and can serve as a nucleophili general base   protonated form can serve as a general acid   serve a role in stabilizing the folded

 

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  depending on the number of the protons they can give up, we define  and triprotic  acids  acids monoprotic , diprotic  and   monoprotic , (e.g. acetic acid or ammonium) have only one dissociable group  (carbonic acid, bicarbonate,   diprotic  (carbonic glycine) have two dissociable groups and   triprotic  (e.g.  (e.g. phosphoric acid) have three dissociable groups.

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pK  values  values they   In the case of multiple pK  are designated by indices: pK  pK 1, 1, pK  pK 2, 2, pK 3, 3, etc •  For amino acids, the pK  p K 1 constant refers to its carboxyl (-COOH) group •  pK  2 refers to its amino (-NH3) group •  the pK  pK 3 is the pK  p K  value  value of its side chain

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  Both the a-amino group and the acarboxyl group are ionizable •  a-COOH group: 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

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  side chain ionizable groups: only 7 amino acids    Asp, glu   His, lys, arg Cys, tyr  

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structures of proteins

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

 

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

 

 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 another methyl group attached to the side chain  chain  

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 Aromatic AA, 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,  (Tyr, Y) contains a hydroxyl   Tyrosine group at the end of the phenyl ring (makes tyrosine less hydrophob hydrophobic) ic) -is a reactive group (the side chains so far have all been unreactive)

accept or donate proteins at physiological pH. For this reason histidine is often found at the active site of enzymes. Lysine (Lys;  (Lys; K) has one of the   Lysine 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 

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Sulfur containing  containing    Cysteine (Cys; C) sulphydryl group (-SH) is extremely reactive, can form hydrogen bonds and disulphide bridges -even though the – the  –SH SH group can form H bonds the long aliphatic part of the side chain makes it quite hydrophobic 

 

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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, AA, neutral, those which are acidic and those which are basic Basic AA contain 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

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

 

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, (Trp, Tyr, Phe) absorb Phe) absorb light in the near UV region of the spectrum (250-300 mm) Trp has highest molar absorptivity   Trp has followed by Tyr, Tyr, with Phe Phe making  making only a small contribution   Disulfide bonds (between Cys residues in proteins) also absorb in the UV range, but much less than the aromatics 

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Posttranslational modifications of AA Posttranslational side chains  chains    Chemical modifications AFTER biosynthesiss of proteins biosynthesi   Occur a few AA residues in some proteins 

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 AA   20 (biologically essential)   Can synthesize 12 -amphibolic -amphibol ic intermediates of glycolysis and CAC (9) -3 (cysteine, tyrosine, hydrolysine) 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 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 (required for the young young but not for adult) Isoelectric point (pI) •  pI = the pH exactly halfway between the two pKa values surrounding 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 negative   (more - than + charges)

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

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

 

Threonine (Thr; T) neutral AA which Threonine (Thr; has a highly reactive (and highly hydrophilic) hydrophili c) 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

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

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

the remaining H+ attaches itself to the weak base, giving weak acids as one of the products  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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pI

  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 charges = the sum of the neg charge  –   Region of buffereing : pKa +/- 1  – 

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

 Acid+Base  Salt+water 

 

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 pK3

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(-1 and -2)

pI = pK1 + pK2/2 = 2.1 + 3.9 = 3

 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 from solution -Acid: substances that can donate a H ion Model    Lewis Model  -acid: accepts a pair of electrons -base: donates a pair of electrons 

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

 

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=pK 1 +pK 2

= 3.2    At pH 2000 4. Nutrient and storage •  Ovalbumin •  Ferritin •  Gliadin •  zein 5. Contractile or motile  –    Actin and myosin myosin 6. Protective  –   Ig, fibrinogen, thrombin 7. Hormones 8. Toxins 9. Receptors Classification Classificati on 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, 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 solubility in water  –   amphoteric

solubility •  Depends on the AA components on the surface  –   High hydrophobic hydrophobic on the surface : low solubility  –   Charged, polar surface residues increase solubility Precipitation: “salting out”   Precipitation: “salting •   Addition of neutral neutral salt (NH4SO4)  –   Increased reaction of charges of protein with the salt and exposes hydrophobic hydrophob ic areas on prtein surface which aggregate and precipitate Coagulation point 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 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 well-defined •  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 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: residues: replace the suff suffixes ixes with – with –yl 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 ionizable in protein protein or polypeptide:   In side chain (7) -  Basic: Histidine, arginine, lysine glutamate -   Acidic: Aspartate, glutamate -  Thiol: Cysteine tyrosine -    Terminal amino acids Secondary structure : •  Due to formation formation of H bonds 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 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 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 chain i is a R-handed helical conformatio conformation n    Each turn of the helix comprises 3.6 amino acids   stiff, rod-like structures   backbone

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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 isoleucine causes steric   Group of isoleucine hindraance due to bulky side ch chains ains small R which allows   Glycine due to small movement destabilize destabilize due to   Group of acidic AA destabilize negative charges which repel each other   Lysine, Arg. Serine, threonine

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  Many alpha helices have predominantly hydrophobic hydrophob ic R on one side and predominantly predominan tly hydrophilic on t he 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) strand) is a stretch of polypeptide chain typically typically 3 to 10 aminoacids long with backbone in an almost fully extended conformation •  β sheet   refers to an assembly  of  refers  of at least two such β strands that are hydrogenhydrogenbonded (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 right-hand ed 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 hydrophil ic residues and are often binding sites

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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 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 -  larger sequence which    A loop is a much larger changes the direction of t he 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

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  Proline and glycine are known as "helix breakers"" because they disrupt the breakers regularity of the α helical backbone conformation -  have unusual c onformational abilities -  commonly found in turns •   Amino acids that prefer prefer to adopt helical conformations in proteins : methionine, alanine, leucine, glutamate and lysine •  prefer to adopt β-strand β -strand conformations -  the large aromatic residues (tryptophan, tyrosine and phenylalanine) -  Cβ Cβ-branched -branched amino acids (isoleucine, valine, and threonine)

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supersecondary structure  supersecondary structure  •  formed by the combi of several neighbouring neighbouri ng 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 super-second ary 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. •

structure is the "global" folding   Tertiary 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 domain is  is the unit of tertiary structure •  Bonds that stabilize tertiary level Hydrophobic   Ionic   H bonds   •

  disulffide   controls the basic function of the protein

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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) Hydrophobicc (between non-polar non-polar Rs)   Hydrophobi disulfide e bonds between cysteine   disulfid

  units of tertiary structure aggregate to form homo- or hetero- multimers •  Hetero-multimers Hetero-multimers     different tertiary domains aggregating together to form a unit •  Homo-multimers Homo-multimers     more common to find copies of the same tertiary same  tertiary domain associating non-covalently •

Separating proteins •  Size -  Ultrafiltration -  centrifugation -  Size exclusion chromatograph chromatography y chromatography y   through a chromatograph 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 chromatography -  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 chromatography   and electrophoresis electrophoresis -  In  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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residues in different polypeptide chains 8

 

  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 aminopeptidase

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  C terminal (hydrazinolysis) sis) and -   Akabori reaction (hydrazinoly 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, hydroxylam ine, and heating an acidic solution Cyanogen bromide specifically   attacks methionine •   Apply Edman degradation degradation to the fragments •

Collagen •  Main protein in CT of animals •  Most abundant protein in mammals (2535%) •  Tropocollagen Tropocollagen:: fundamental unit of collagen (3 chains) •   Alpha helix : L-handed L-handed helix •  3 entwine to form R-handed triple helix -Resists unwinding: 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

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Type I •  is the most common fibril-forming collagen (90%) •  Its fibrils make up the mineralized matrix in bone 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-form fibril-forming ing collagen in cartilage   cartilage •   Vitreous body, nucleus nucleus pulposus 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 hydroxyprol hydroxyproline ine 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) (preprocollag en) (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 hydroxylys ine linkage

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Disorders Ehlers Danlos syndrome -  Faulty collagen synthesis Hyperextensible e skin   Hyperextensibl   Easy bruising   Hypermobile joints •   Autosomial dominant dominant or recessive recessive •  Type III most frequently affected

hemoglobin •  Metalloprotein (iron) •  oxygen-transp oxygen-transport ort 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 t otal 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 abundant in the proteins of hair, hooves, and the keratin of the skin

Osteogenesis imperfecta Osteogenesis •  Brittle bone disease •   Autosomal dominant dominant , most common form •   Abnormal type type I ; (type II :fatal utero)   Multiple fractures (during birth)   Blue sclerae (translucent connective tissue over choroid)   Hearing loss (abn middle ear bones) dentin – dental  dental abn   Lack dentin –  Alport’s sundrome sundrome •   Abnormal type type IV •  (basement membrane of kidney, ears, eyes) •  Most common form X-linked recessive •  Nephritis, deafness, ocular  Vit C deficiency deficiency •  Prolyl and lysyl hydroxylases: poor function, therefore no cross links

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Myoglobin •  a single-chain globular protein of 153 aa, containing a heme (Fe-containing porphyrin)) prosthetic grp in the center porphyrin around wc remaining apoprotein folds •  8 alpha helices and a hydrophobic core •  primary oxygen-carrying pigment of muscle tissues. 9