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					Basics of protein structure and
stability III: Anatomy of protein
              structure
      Biochem 565, Fall 2007
            08/29/08
             Cordes
      The backbone conformation of proteins:
    combination of regular and irregular structures
      human
      hexokinase
      type I
      (1QHA)
      102 kDa
      Rosano et al.
      Structure 1999.


      pig insulin
      (1ZNI)
      5.7 kDa
      Bentley et al.
      Nature 1976.

These “ribbon” diagrams show the “skeleton” of a protein. They are a smoothed
representation of the “backbone” or “main chain” structure and do not show the side
chains
Backbone or main-chain conformation
             O                        O                        O            O

                     H                        H                    H
H2N    CH    C       N       CH       C       N       CH       C   N   CH   C   O
                                                                                H

       R1                    R2                       R3               R4



                                                 
                                                        

                 residue 2                    residue 3

There are three bonds between “main chain” atoms (everything but
the side chain) per residue, and torsional rotation can occur about any
of these bonds, in principle. Hence, each residue has 3 angles that
describe the main chain conformation for that residue.
         Backbone conformation:
   resonance forms of the peptide group
                    O
                             ..   R'       –O            R'
                                                     +
                        C    N                  C    N
                    R             H         R            H


The delocalization of the lone pair of the nitrogen onto the carbonyl oxygen
shown in the resonance form on the right imparts significant double bond
character (40%) to the peptide bond.

Breaking of this double bond character by rotation of the peptide bond requires
on the order of 18-21 kcal/mol.

Consequently there is not free rotation around the peptide bond: rotation about
the peptide bond happens on the time scale of seconds/minutes--very slow
                     Consequences of double bond
                     character in the peptide bond
           1.24 Å                   H
                     O
                           123.2°            R
           121.1°
                               121.9°
                                                           the peptide C-N bond is 0.12A
H2N                  C                  C            OH   shorter than the Calpha-N bond.
            C        1.33 Å    N    1.45 Å                and the C=O is 0.02A longer than
                                                           that of aldehydes and ketones.
       R        H              H                 O




                                    H
                      O
                                             R
                                                          All six of the atoms highlighted at
H 2N                   C                C            OH
                                                          left lie in the same plane, and as
                                                          with carbon-carbon double bonds
             C                  N
                                                          there are two configurations--cis
       R
                                                            and trans (trans shown at left)
                 H              H                O
        Consequences of double bond
        character in the peptide bond
       trans peptide bond                         cis peptide bond




Still another consequence: in the cis form, the R groups in adjacent residues
  tend to clash. Hence almost all peptide bonds in proteins are in the trans
                               configuration.
  ...and that means that the dihedral angle describing rotation around the
peptide bond, defined by the four atoms Ca(i)-C-N-Ca(i+1), will generally be
         close to 180°. This angle is known by the greek symbol .

                                          H
                               O
                                                    R

                                     
            H2N                C              Ca              OH

                      Ca              N



                  R        H          H                 O



                  residue i               residue i+1

   So the properties of the peptide bond place a strong restriction on the
backbone conformation or main-chain conformation of proteins, that is to
         say, the spatial configuration of the non side-chain atoms.
The peptidyl-proline bond
               O            H                           O       cis peptidyl-proline
                       Ca
                                N
                   N                                        N
         R H                O                     R H                   H
                                                         Ca
                                                                    N
         trans peptidyl-proline
                                                            O
• The peptidyl proline bond is an exception.
• It can be in either the trans or cis configuration, and the equilibrium constant
favors the trans only very slightly.
 • Roughly 20% of all peptidyl proline bonds in native proteins are in the cis
configuration.
• This is in part because the amide hydrogen is replaced by a methylene
group, which can clash with the R group of the preceding amino acid.
• Remember that there is still a kinetic effect on the rate of isomerization of the
peptidyl proline bond that is similar to that for other peptide bonds--proline cis-
trans isomerization can take seconds/minutes to occur, and this can actually
limit the rate at which a protein adopts its “native” configuration beginning from
a disordered structure (more on this later).
Backbone or main-chain conformation
             O                      O                 O            O

                   H                     H                H
H2N    CH    C     N       CH       C    N       CH   C   N   CH   C   O
                                                                       H

       R1                  R2                    R3           R4



                                            
             ~ 180°                    ~ 180° 

                 residue 2               residue 3


So, one of the three degrees of freedom of the protein backbone is
essentially eliminated by the properties of the peptide bond. What
about the other two?
  carboxy (C) terminal end                    and  angles
Ri+1                        peptide planes
           Cai+1
  H

                   Ni+1      Hi+1
                                    Torsional angles are defined by
 Oi        C' i
                             H
                                    four atoms:
          i                              --> Ni-Ca- C’i -Ni+1
                   Cai
           i                Ci           --> C’i-1-Ni-Ca- C’i
 Hi        Ni
                                    Notice that these are defined
                   C' i-1    Oi-1
                                    from N to C terminus, using
Ri-1
                                    main chain atoms only.
           Cai-1

      H
                                    A residue’s conformation is usually
                                    listed as (,), since  is close
                                    to 180 for almost all residues.
amino (N) terminal end
Unlike the peptide bond, free rotation occurs about the other two backbone bonds, but
    steric interactions within the polypeptide still severely limit plausible conformations, so
    that only certain combinations of phi and psi angles are “allowed”


                                                     and between carbonyls



                                                                   or combinations of
                                                                  an R group, carbonyl
        between R groups                                            or amide group.


                      180


                                                                         not allowed
                       0
 allowed


                     -180
                            -180           0               180
   Steric clashes disallow some  and 
               combinations

                                                   Theoretical calculations using hard
                                                   sphere approximations suggest which
                                                   phi and psi combinations cause
                                                   clashes, and between which atoms.

                                                   Cross-hatched regions are “allowed”
                                                   for all residue types. The larger
                                                   regions in the four corners are
                                                   allowed for glycine because it lacks a
                                                   side chain, so that no steric clashes
                                                   involving the beta carbon are
                                                   possible.


from web version of JS Richardson’s “Anatomy and
Taxonomy of Protein Structure”
http://kinemage.biochem.duke.edu/~jsr/index.html
Observed  and  combinations
         in proteins
                Phi-psi combinations actually
                observed in proteins with known
                high-quality structures.

                Gly residues are excluded from
                this plot, as are Pro residues and
                residues which precede Pro
                (more on this later).

                Contours enclose 98% and 99.95%
                of the data respectively.

                Notice that the observed conformations
                do not exactly coincide with the
                theoretically allowed/disallowed confs
                based on steric clashes.

                                 from Lovell SC et al.
                                 Proteins 50, 437 (2003)
Sample “Ramachandran plot” for a
           protein red= allowed
                    yellow= additionally allowed
                    pale yellow= generously allowed
                    white= disallowed


                    The squares denote non-glycine
                    residues, while the triangles are
                    glycines. Glycines have no side
                    chain and are not as restricted
                    because of the lack of side chain
                    steric clashes.

                     This protein has 219 residues,
                     90% of which are in the “allowed”
                     region and 10% of which are
                     in “additionally allowed” regions.
                     None are in the other regions
                     (except glycines,
                     which don’t count)

				
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posted:4/26/2011
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