? Structure organization of the Proteins:
1) Primary structure
2) Secondary Structure
3) Tertiary Structure
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4) Quaternary StructureProperties of Amino Acids
Amino Acids have an Asymmetric Center
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? Optically active molecules have an asymmetry such that they are notsuperimposable on their mirror image.
? C atoms of all aa are asymmetric centers and optically active except glycine, in
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which R=H two of the four substituents on the -carbon atoms are hydrogen.
? C is a chiral center, this carbon atom is attached to four different groups.
Absolute Configuration of Amino Acids
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? All -amino acids (aa) derived from
proteins have the L-stereochemical
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configuration.? They all have the same relative
configuration about their C atoms.
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? If the NH +
3 projected to the left, the aa
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has L (Levo) absolute configuration.? NH +
3 projected to the right, the aa has
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Fig. 2.7. Textbook of Biochemistry with Clinical Correlations, 4th edition by
Thomas M Devlin
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D (Dextro) absolute configuration.Acid-Base Properties of Amino Acids
? Amino acids in aqueous solution, contain weakly acidic -carboxyl groups and
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weakly basic -amino groups.? Charged and uncharged form of the ionizable weak acid groups ?COOH and -NH +
3
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exist in protonic equilibrium:
R-COOH
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R-COO- + H+R- NH3+
R- NH2+ + H+
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? Both R-COOH and R-NH + are weak acids. R-COOH are stronger acid than R-NH +
3
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3? At physiological pH, carboxyl group exists as R-COO- and amino group as R-NH3+
Henderson-Hasselbalch equation: Quantitative relationship between pH and
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concentration of a weak acid (HA) and its conjugate base (A-).
Derivation of Henderson-Hasselbalch equation: Consider the release of a proton
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by a weak acid represented by HA:The "salt" or conjugate base (A?) is the ionized form of a weak acid.
? Dissociation constant of the acid, Ka, is:
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OR? By taking the negative logarithm of both sides:
? Substituting pH = -log[H+] and pK a = -log K a obtain Henderson-Hasselbalch
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equation:
Cont--
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? Larger the K a, the stronger the acid, because most of the HA has dissociated intoH+ and A?.
? Conversely, the smaller the K a, the less acid has dissociated and, therefore, the
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weaker the acid.
? pKa values for a particular molecule are determined by titration.
Titration of an Amino Acids
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Dissociation of the carboxyl group of
Alanine:
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Ionic forms of alanine in acidic, neutral, and basic solutions? At a low (acidic) pH, both of
ionizable -carboxyl and -amino
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group are protonated.
? As pH of solution raised, ? COOH
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group of form I can dissociate bydonating a proton to the medium.
? Release of a proton results in the
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formation of carboxylate group, ?
Fig. 1.10: Lippincott's il ustrated reviews- Biochemistry 6th edition
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COO? of form I , which is the dipolarform of the Alanine.
Cont--
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Dissociation of the amino group:
? Second titratable group of alanine is amino (? NH3+) group.
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? This is a much weaker acid than the ? COOH group and, i.e, has a much smallerdissociation constant, K 2.
? Release of a proton from protonated amino group of form I results in fully
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deprotonated form of alanine, form I I.
Titration Curve of Alanine
? pKa for acidic group (?COOH) is pK 1 is 2.35,
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whereas pKa for next acidic group (? NH3+)
is pK 2 is 9.69.
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? Isoelectric pH (pI) of Alanine is:Fig. 1.11: Lippincott's il ustrated reviews- Biochemistry 6th edition
Cont--
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? In clinical laboratory, knowledge of the isoelectric pH guides, selection of
conditions for electrophoretic separations.
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? For example, two simple aa (with one COOH and one NH3+ group) separated byelectrophoresis either at an acidic or basic pH that exploits subtle differences in
net charge based on subtle differences in pK 1 or pK 2 values.
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Ultraviolet Spectra of Ty r, Phe andTrp? Amino acids do not absorb visible light.
? Ty r, Phe, and Trp absorb high-wavelength
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(250-290nm) UV light.
? Absorption of light at 280nm used to
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estimate the concentration of a protein insolution, if the number of Trp and Ty r
residues in the protein is known.
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Fig.3.7. Harper's Il ustrated Biochemistry 30th edition
Structural organization of Proteins
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Fig.3.16. Lehninger Principles of BiochemistryPrimary Structure of Proteins
Primary Structure
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? Amino acids are linked by peptide bonds to form polypeptide chains.? Ordered sequence of aa in a polypeptide chains is the primary structure of the
protein.
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? It is the unique primary structure that enables a polypeptide chain to fold into a
specific 3-D structure that gives the protein its chemical and physiological
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properties.Amino Acids are Polymerized into Peptides and Proteins
? -carboxyl group of an aa with side chain R1
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forms a covalent peptide bond with -aminogroup of the aa with side chain R2 by elimination
of a molecule of H 2O.
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? Dipeptide (contain two aa bonded to each other
via a single peptide bond).
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Fig. 2.8. Peptide bond formation:Textbook of Biochemistry with Clinical Correlations
4th edition by Thomas M Devlin
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Cont--
? Tripeptides (contains three aa) form a second peptide bond through its terminal
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carboxylic acid group and the a amino of a third amino acid (R3).? Repetition of this process form a polypeptide or protein of specific aa sequence
(R1R,2R,3R4 Rn).
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Components of a Polypeptide Chain? It consists of a repeating part, backbone, and a
variable part, comprising the distinctive side
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chains.
? Polypeptide backbone is rich in hydrogen-
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bonding potential. Each residue contains acarbonyl group, and, with the exception of
proline, an NH group.
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? Mass of a protein expressed in units of
daltons; one Dalton (Da) is equal to one
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Fig.2.15: Components of a polypeptide chain: Biochemistry 7th edition by Berg,atomic mass unit.
Tymoczko and Stryer
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Peptide Bond has Partial Double-Bond Character
? Single bond linked the -carboxyl and -nitrogen
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atoms, this peptide bond exhibits partial double-bond character.
? Bond that connects a carbonyl carbon to an -
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nitrogen cannot rotate and thus conformation of
the peptide backbone is constrained.
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? Almost all peptide bonds in proteins are trans.Fig.2.18: Peptide bonds are planar: Biochemistry 7th edition by Berg, Tymoczko
and Stryer
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? The O, C, N, and H atoms of a peptide bond are
coplanar.
Rotation about Bonds in a Polypeptide
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? Structure of each aa in a polypeptide adjusted by
rotation about two single bonds.
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? Phi () is angle of rotation about bond betweenN-C bond.
? psi () is angle of rotation about bond between
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C -C
bond.
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? Freedom of rotation about two bonds of each aaFig.2.22: Biochemistry 7th edition by Berg, Tymoczko and Stryer
allows proteins to fold in many ways.
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Ramachandran Plot
? In principle, values of and bet. -180?
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and +180?, but many values are prohibitedby steric interference between atoms in
the polypeptide backbone and aa side
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chains
? Allowed/favorable regions with no steric
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overlap are shown in dark green;borderline regions are shown in light
green.
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? In peptides, for aa other than glycine, most
combinations of and angles are
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Fig.2.23: Biochemistry 7th edition by Berg, Tymoczko and Stryerdisallowed because of steric hindrance.
Amino Acid Sequences have Direction
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? A polypeptide chain has polarity because itsends are different: an -amino group is
present at one end and an -carboxyl group
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at the other.
? Amino end is taken to be the beginning of a
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polypeptide chain, and so the sequence of aain a polypeptide chain is written starting with
the amino-terminal residue.
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Fig.2.14: Amino acid sequences have direction: Biochemistry 7th edition by
Berg, Tymoczko and Stryer
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Cross-Links: Formation of a Disulfide Bond? In some proteins, the linear polypeptide
chain is cross-linked.
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? The most common cross-links are disulfide
bonds, formed by the oxidation of a pair of
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cysteine residues.? The resulting unit of two linked cysteines is
called cystine.
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Fig.2.16: Cross-links: Biochemistry 7th edition by Berg, Tymoczko and Stryer
Cont--
? Extracellular proteins often have several disulfide bonds, whereas
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intracel ular proteins lack them.
? For ex., collagen fibers in connective tissue and fibrin blood clots.
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Summary? All aa possess at least two weakly acidic functional groups, R-NH3+ and R-COOH.
? Many also possess additional weakly acidic functional groups such as phenolic -
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OH, -SH, guanidino, or imidazole moieties.
? The pKa values of all functional groups of an aa or of a peptide dictate its net
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charge at a given pH.? pI, the isoelectric pH, is the pH at which an aa bears no net charge, and does not
move in a direct current electrical field.
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Cont--? The most important element of primary structure is the sequence of amino acid
residues.
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? Nature of the covalent bonds in the polypeptide backbone places constraints on
structure.
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? Peptide bond has a partial double bond character that keeps the entire peptidegroup in a rigid planar configuration.
? The N-C and C -C
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bonds can rotate to assume bond angles of and ,
respectively.
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Group Discussion? Subtopics of previous class discussed in groups.
Reference Books
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1) Harper 's Il ustrated Biochemistry-30th edition.2) Textbook of Biochemistry with Clinical Correlations. 4th edition. Thomas M.
Devlin.
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3) Biochemistry. 4th edition. Donald Voet and Judith G. Voet.4) Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer.
5) Lehninger Principles of Biochemistry.
31
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