Protein structure - Pharmaceutical Biotechnology: Concepts and Aapplications - page 21

Planar (rigid) peptide bonds

H O R 2 H H

N C C N C C N C

α α α

H R 1 H H O R 3

C - C bond free to rotate,

angle of rotation =

α

N - C bond free to rotate,

angle of rotation = φ

ψ

α

Figure 2.3 Fragment of polypeptide chain backbone illustrating rigid peptide bonds and the intervening
N ! C α and C α ! C backbone linkages, which are free to rotate

Whereas the peptide bond is rigid, the other two bond types found in the polypeptide backbone
(i.e. the N!C α bond and the C α !C bond, Figure 2.3) are free to rotate. The polypeptide back-
bone can thus be viewed as a series of planar 'plates' that can rotate relative to one another. The
angle of rotation around the N!C α bond is termed φ (phi) and that around the C α !C bond is
termed (psi) ( Figure 2.3). These angles are also known as rotation angles, dihedral angles or

torsion angles. By convention, these angles are defi ned as being 180 when the polypeptide chain is
in its fully extended, trans form. In principle, each bond can rotate to any value between

ψ

and
180 . However, the degrees of rotation actually observed are restricted due to the occurrence of
steric hindrance between atoms of the polypeptide backbone and those of amino acid side chains.
For each amino acid residue in a polypeptide backbone, the actual φ and ψ angles that are physi-
cally possible can be calculated, and these angle pairs are often plotted against each other in a dia-
gram termed a Ramachandran plot. Sterically allowable angles fall within relatively narrow bands in
most instances. A greater than average degree of

180

rotational freedom is observed around glycine
residues, due to the latter's small R group - hence steric hindrance is minimized. On the other hand,
bond angle freedom around proline residues is quite restricted due to this amino acid's unusual
structure ( Figure 2.1). The φ and ψ angles allowable around each C α in a polypeptide backbone obvi-
ously exert a major infl uence upon the fi nal three-dimensional shape assumed by the polypeptide.

φ / ψ

2.2.3 Amino acid sequence determination

The amino acid sequence of a polypeptide may be determined directly via chemical sequencing or by
physical fragmentation and analysis, usually by mass spectrometry. Direct chemical sequencing was
the only method available until the 1970s. Insulin was the fi rst protein to be sequenced by this approach
(in 1953), requiring several years and several hundred grams of protein to complete. The method has
been refi ned and automated over the years, such that, today, polypeptides containing 100 amino acids or
more can be automatically sequenced within a few hours, using microgram to milligram levels of pro-
tein. The actual chemical sequencing procedure employed is termed the Edman degradation method.