the array surface for a period of time, then washed off, and the position(s) where the
protein has bound, identified (see below). Since it is known which protein was immo-
bilised in each position of the chip, each pair of interacting proteins can be identified.
Saccharomyces cerevisiaeagain provides a good example of the successful use of
this technology where a protein array was used to identify yeast proteins that bind to
the protein calmodulin (an important protein involved in calcium regulation). Five
thousand eight hundred yeast ORFs were cloned into a yeast high copy expression
vector, and each of the expressed proteins purified. Each protein was then spotted
at high density onto nickel-coated glass microscope slides. Since each protein also
contained a (His) 6 -Tag (which binds to nickel) introduced at the C terminus, proteins
were attached to the surface in an orientated manner, the C terminus being linked to the
nickel-coated glass through the (His) 6 sequence, while the rest of the molecule was
therefore suitably orientated away from the surface of the array to be available for
interaction with another protein. The array was then incubated in a solution of calmo-
dulin that had been labelled with biotin. The calmodulin was then washed off and the
positions where calmodulin had bound to the array were identified by incubating the
array with a solution of fluorescently labelled avidin (the protein avidin binds strongly
to the small-molecular-mass vitamin biotin: see Section 10.3.8). The use of ultraviolet
light thus identified fluorescence where the screening molecules had bound. In total,
33 new proteins that bind calmodulin were discovered in this way.
Figure 8.9 (see also colour section) shows an interaction map of the yeast proteome.
The authors constructed the map from published data on protein–protein interactions
in yeast. The map contains 1584 proteins and 2358 interactions. Proteins are coloured
according to their functional role, e.g. proteins involved in membrane fusion (blue),
lipid metabolism (yellow), cell structure (green), etc. If one views the electronic
version of this publication it is possible for the reader to zoom in and search for
protein names and to read interactions more clearly.
Figure 8.10 (see also colour section) is a summary of Fig. 8.9 showing the number of
interactions of proteins from each functional group with proteins of their own and
other groups. The word function means the cellular role of the protein. Numbers in
parentheses indicate, first, the number of interactions within a group and, secondly,
the number of proteins within a group. Numbers on connecting lines indicate the
numbers of interactions between proteins of the two connected groups. For example,
in the upper left-hand corner, there are 77 interactions between the 21 proteins
involved in membrane fusion and 141 proteins involved in vesicular transport.
Looking at the bottom right of the diagram it can be seen that some proteins involved
in RNA processing/modification not surprisingly also interact with proteins involved
in RNA turnover, RNA splicing, RNA transcription and protein synthesis.8.5.6 Systems biology
It can be seen from the section on proteomics that the study of proteins is moving
away from methods that involve the purification and study of individual proteins.
Nowadays proteins are more likely to be studied as a stained spot on a complex 2-D
gel pattern, often present in as little as nanogram amounts, more often than not using349 8.5 Proteomics and protein function