BioPHYSICAL chemistry

being studied by many directions in research. For example, the response
of every gene of an organism to environmental changes is being invest-
igated. Every gene encodes a protein that has a specific function in the cell.
Proteomics is the study of how every protein of an organism responds
to changes. Although the main characteristic of a gene is its nucleotide
sequence, an understanding of the properties of a protein requires identi-
fication of not only the protein sequence, but also of how the polypeptide
chain folds as a three-dimensional object. Therefore a major goal of pro-
teomics is to determine the three-dimensional structures of every protein
within certaintargeted organisms.
The technique of X-ray diffraction is the primary tool used by scientists
to determine the three-dimensional structure of proteins. Many aspects
of this technique have been optimized as described in Chapter 15. The
major limitation in most cases is that the technique requires a crystallized
protein. Since proteins can assemble into complexes and undergo post-
translational modifications, the number of proteins per organism is not
uniquely determined by the genome, but certainly is in the range of tens
of thousands. To determine every protein structure within a reasonable
time, it is necessary to apply automated, high-throughput methods. Despite
these improvements, the crystallization of any given protein may take years,
largely because the thermodynamic properties that lead to crystallization
remain difficult to apply and crystallization proceeds using a trial-and-error
approach involving thousands of conditions (Kam et al. 1978; McPherson
et al. 1995).
Proteins need to always be in the proper buffering conditions and ionic
strength. Crystallization represents changing the state of the protein by
its removal from a solution into a crystalline array. Thus, there is a com-
petition between the protein in liquid solution and in the crystalline state.
The protein in solution will have a certain free energy (Figure 4.17). If the
protein forms a microcrystal consisting of only a few proteins, the free energy
will increase. The favorable introduction of additional bonds between
proteins cannot overcome the penalty of the decreased entropy. Thus, the
monomer state is favored over the microcrystalline state. Only when the
crystal reaches a certain critical size does the
free energy decrease with increasing size,
hence favoring the crystallization process. In
practice, if the energy increase due to micro-
crystalline formation is low, then the system,
after a period of time, can form a crystal of
the critical size and the crystallization can pro-
ceed. While this may seem straightforward,
the problem is that proteins can also form
another state, a disordered precipitate, that
is energetically favorable to growing large
crystals, and this can deplete the supply of

CHAPTER 4 PHASE DIAGRAMS AND MIXTURES 89

Quasiequilibrium

distribution of aggregates

Ordered (crystals)

Critical size

V

Disordered

(amorphous precipitation)

0

Free energy

Aggregate size

Postnucleation

growth

Figure 4.17

Dependence of the

Gibbs energy on the

size of an aggregate.

Modified from Kam

et al. (1978).