Proteomics

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Proteomics
Session 2 protein structure
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Outline

• Proteins are built from a repertoire of 20 amino
  acids
• 1. Primary Structure Amino acids are linked by
  peptide bonds to form polypeptide chains
• 2. Secondary Structure a helix, b sheet, and
  turns and Loops
• 3. Tertiary Structure Folding of proteins
• 4. Quaternary Structure Multi-subunit
  Structures
• The Amino Acid Sequence of a Protein Determines
  Its Three-Dimensional Structure

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Levels of structure in proteins
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Key Properties of proteins

• 1. Proteins are linear polymers built of monomer
  units called amino acids. The function of a
  protein depends on its 3D structure (Fig 3.1).
• 2. Proteins have various functional groups this
  enables them to have various functions.
• 3. Proteins can interact with one another and
  with other biological macromolecules to form
  complex assemblies.
• 4. Some proteins are quite rigid some are
  flexible (Fig 3.2)

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Fig 3.1 3.2
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1. Primary structure

• Amino acid is dipolar ions, the ionization state
  of it depends on the pH of the medium.

• Only L-amino acids
• found in proteins (arbitrary selection of
  L-over D-form.)

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Review on Amino Acids

• Building blocks for proteins
• There are 20 of them
• Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gly
  (G), Gln (Q), His (H), Glu (E), Ile (I), Leu (L),
  Lys (K), Met (M), Phe (F), Pro (P), Thr (T), Tyr
  (Y), Trp (W), Ser (S) and Val (V)
• Need to know 3 letter symbols, the one letter
  symbol is also given to each aa.
• Essential amino acidsPVT TIM HALL (Phe, Val,
  Thr, Trp, Ile, Met, His, Arg, Lys, Leu)

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Amino acids with aliphatic side chains
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Sulphur containing amino acids
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-OH containing amino acids
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Aromatic amino acids
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Positively charged amino acids
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Negatively charged and Uncharged amino acids
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Beers law

• A compounds extinction coefficient indicates its
  ability to absorb light.
• Beers law AecL
• E--gtext. coefficient
• C--gtconcentration
• L---gt Length through which light passes

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The importance of understanding amino acid
sequence

• To understand proteins function
• To understand the rules which are important in
  folding of polypeptide chains
• Part of molecular pathology
• Reveals many information about its evolutionary
  history

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Peptide bonds
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The peptide unit is rigid

• Peptide unit is not free to rotate, partial
  double bond character!
• The hydrogen of the substituted amino group is
  nearly always trans (opposite) to the oxygen of
  the carbonyl group.
• The bond between the carbonylcarbon and the
  nitrogen atom of the peptide unit has partial
  double-bond character!
• There is a larger degree of rotational on either
  side of the peptide bond.

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Fig 3.3
Fig 3.4
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Disulfide bonds
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Two conformations are possible

• 1. Trans
• 2. Cis
• Almost all peptide bonds in proteins are in trans
  configuration!
• This preference for trans over cis can be
  explained by the fact that steric clashes between
  groups prevent cis configuration.

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Freedom of rotation

• There is a freedom of rotation Nitrogen-aC, and
  carbonyl-aC.
• This freedom of rotation allows proteins to fold
  in many different ways!
• phi The angle of rotation about the bond
  between the amino nitrogen and the aC
• psi The angle of rotation about the bond
  between the aC and the carbonyl carbon

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Rotation of peptide bonds and ramachandran plot
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2. Secondary structure

• a-helix
• b-sheet
• Turn and loop

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a- helix

• Rod like structure
• Stabilized by H bonds (CO of n forms H bond with
  NH of n4)
• There is 3.6 aa residues per turn of helix,
• a-helical content of proteins ranges,
• _at_ Mb and Hb 75
• _at_ chymotrypsin almost none

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General illustrations of a-helix
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3-D Structural illustration for a-helix
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Distribution of a-helix structure on ramachandran
plot
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b- sheet

• It is fully extended structure
• The distance between
• 2 aa--gt 3.5 A (1.5 A in a-helix)
• The side chains can run in opposite or same
  directions
• 2 polypeptide chains are held by H bonds

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Anti-parallel beta sheet
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Parallel beta sheet
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3-D Structural illustration for b-sheet
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Distribution of b-sheet structure on ramachandran
plot
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Pp chains can change direction by making reverse
turns

• Most proteins have compact globular shape, how?
• By reverse turns
• CO group of residue x is H-bonded to the NH of
  residue x3 to stabilize the turn. (fig3.5)
• More complicated turns are called LOOPS
• They do not have regular structure

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Fig 3.5
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Tertiary structure

• Proteins fold into compact structures with
  nonpolar cores
• Myoglobin surface has many charged as well as
  some hydrophobic
• The overall shape of a protein -----gt tertiary
• Domains Some polypeptide chains fold into two or
  more compact regions that are connected by a
  flexible segment (30-400 aa)

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Myoglobin structure
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Myoglobin structure contd.
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Myoglobin in solution
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Three dimensional structures of cytochrome c,
lysozyme and ribonuclease
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Collagenthe triple stranded helix

• Means glue
• Most abundant protein in mammals
• Aa sequence is very regular. Every third amino
  acid is Gly
• Gly-X-HydroxyPro
• No H bond within the strand
• 3 strands wind around each other to form a
  super-helical cable
• Human genetic defects
• Osteogenesis imperfecta
• Ehlers-Danlos Syndrome

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Collagen structure
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Quartenary Structure

• Polypeptide chains can assemble into
  multi-subunit structures
• If there is more than 1 pp chain then those
  proteins can exhibit a 4th level of structural
  organization
• Quartenary structure refers to the spatial
  arrangement of subunits and the nature of their
  interaction

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Two subunits
Four subunits
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The amino acid sequence of a protein determines
its 3D structure

• The classic work of Christian Anfinsen
• Ribonuclease has 124 a.a. and crosslinked by 4
  S-S
• These experiments showed that the information
  needed to specify the catalytically active
  structure of ribonuclease is contained in its
  AMINO ACID SEQUENCE!
• Similar refolding experiments done with other
  proteins (small proteins)
• Some proteins refolding does not take place
  efficiently! (big proteins)

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Ribonuclease
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Reductant and reduction
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Strong denaturant
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Denaturation of ribonuclease
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Renaturation of ribonuclease
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What determines alpha, beta or turns?

• Ala, Glu, Leu ---gt a-helix
• Val, Ile ---gt b-sheet
• Pro ---gt turns
• Reasons alpha is a default conformation
• Val, Ile, Thr ---gtdestabilize alpha helix
• Ser, Asp, Asn ----gt disrupt alpha because their
  side chains are H-bond donor or acceptor that may
  interact with the main chain H-bond in alpha
  helix.

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Hydrogen donor or acceptor
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Continue

• Pro---gt tends to disrupt both a-helix and b-sheet
  because it lacks an NH group to make H-bonds. NH
  is involved in ring structure.
• Gly---gt smallest amino acid, it fits into all
  structures for this reason it does not favor any
  structure.

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Many proteins can adopt alternative conformations
in different proteins

• VDLLJKN--gt a-helix
• Or the same peptide could make b-sheets in
  another protein!
• Prion disease (mad cow disease) results when a
  protein called prion converts from its normal
  configuration to an altered one this altered one
  forms large aggregates.

VDLLJKN
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Protein folding is a highly cooperative process

• Proteins can be denatured by heat or by
  chemicals.
• Unfolding is a rather sharp transition like all
  or none kind which results from a cooperative
  transition.
• During transition, there is a 50/50 mixture of
  fully folded and fully unfolded proteins.

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Protein modification and cleavage confer new
capabilities

• Proteins have various functions relying solely on
  these 20 different amino acids.
• Many proteins are covalently modified also to
  increase their functions!
• Glycosylation
• Acetyl attachment
• Hydroxy addition
• Carboxyglutamate
• Phosphorylation

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Examples of covalent modifications
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Addition of special groups

• Certain jellyfish produce a fluorescent green
  protein (GFP)
• Source of fluorescent is made by spontaneous
  rearrangement and oxidation of the sequence
  Ser-Tyr-Gly within the center of proteins.
• Researchers use this protein as a marker.

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Green fluorescent protein, GFP