Page 156 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Encyclopedia of Physical Science and Technology EN013D-616 July 27, 2001 12:5

196 Protein Structure

enzymatically active in the lattice. In others conforma- the underlying physical principles that control the con-
tional changes are observed between the substrate-free formation and function of proteins. This hierarchy also
and substrate-bound forms of the enzyme. Typically this reflects one conceptual view of protein folding, where the
requires the crystallization of site-directed mutant pro- local secondary structural elements form first, followed by
teins complexed with the substrate(s) or the study of com- the tertiary and quaternary structure. The following dis-
plexes with substrate analogs. Except for the use of Laue cussion of protein structure is organized according to the
techniques,proteincrystallographyyieldsatime-averaged preceding hierarchy.
view of the protein structure. Careful analysis of accurate
X-raydiffractiondatamayprovidesomeindicationofcon-
A. Primary Structure
formational flexibility, but that aspect of protein structure
is best suited to spectroscopic techniques such as NMR. The character of a protein is determined by the amino acid
sequence and composition of the polypeptide chain. By
convention the order of amino acids in a protein is listed
B. NMR
starting at the N-terminal and ending at the C-terminal
The use of NMR to determine protein structures is a more amino acid residue. The N-terminal amino acid carries
recent development than X-ray diffraction. It has the ad- a free amino group, whereas the C-terminal residue re-
vantage that the analysis can be performed in the solution tains a free carboxyl group. These terminal residues of
state of the protein which removes any artifacts introduced the polypeptide chain are also referred to as the amino
by crystallization. Its major disadvantage is the size lim- and carboxy terminus of the protein, respectively. Almost
itation, which restricts most analyses to smaller proteins all protein sequences are determined indirectly by DNA
(<40 kDa), although it is anticipated that improvements sequencing. Chemical sequencing, either by automated
in the technology will extend the size limitation. Edman degradation or by mass spectroscopy, is still nec-
Structural studies on proteins became possible with the essary to identify a protein from its original source and
advent of multidimensional NMR techniques. These rely to prove the presence of post-translational modifications.
15
13
on the use of isotopic labeling with C, and N and tech- All sequences of interest should be examined for errors by
1
niquestoprovideafacilemethodforassigningallofthe H resequencing and comparison with orthologous proteins.
resonances in a protein, which would otherwise be a diffi-
cult task. The measurement of nuclear Overhauser effect
B. Conformational Restrictions
(NOE) intensities provide much of the distance informa-
on the Polypeptide Chain
tion necessary to derive a structure, although additional
chemical shift information is needed for a high-resolution The amino acids in a protein are linked by an amide link-
structural determination. age that is referred to as the peptide bond (Fig. 3). There
Once a set of distance information has been obtained are several key features to this bond. It is planar as a
a series of models are generated and optimized by en- consequence of the partial double bond character of the
ergy minimization and molecular dynamics within the carbon–nitrogen bond. It is almost always in the trans
restraints imposed by the distance information. The ad- configuration. The peptide bond is fairly rigid where the
vantage of this approach is that it provides structural in- barrier to rotation is ∼20 kcal/mol. The carbonyl oxygen
formation on the protein in solution, the drawback is that and amide hydrogen carry a partial negative and positive
surface residues and loops appear less well defined be- charge, respectively, which allow each of them to form
cause there are generally fewer distance restraints for these a hydrogen bond. This linkage profoundly influences the
components. The great strength of NMR is that it can yield stability, conformation, structure, and function of proteins.
specific information concerning the pK a of an individual The atoms that form the backbone of the linear poly-
group in a protein as well as providing insight into the peptide chain are usually referred to as the main-chain
dynamical properties of the macromolecule. atoms. By convention the conformational (torsional) an-
gles adopted by the main chain atoms are denoted by φ, ψ,
and ω as shown in Fig. 3. Of these, ω describes the peptide
IV. STRUCTURAL HIERACHY bond and usually adopts a value of 180 . In principle there
◦
is free rotation about the other two angles (φ and ψ); how-
The structure of a protein is generally understood in terms ever, the peptide bond and presence of a β-carbon places
of an organizational hierarchy that consists of protein se- substantial restrictions on these conformational angles.
quence, local secondary structure, tertiary structure, and Conformational energy calculations and experimental
finally quaternary structure. The study of protein struc- observations based on high-resolution X-ray structure de-
ture in these terms has led to a greater understanding of termination show that generally only ∼8% of the possible