BioPHYSICAL chemistry

aided by fusing a GFP-like protein that

fluoresces in the red to the cellular pro-

teins that had been localized previously.

The tagged open reading frames and pro-

teins were examined at different cellular

locations using fluorescence microscopy.

The fluorescence showed that the

expression of the open reading frames

could be organized into distinct subcellu-

lar categories. Possible protein–protein

interactions of the open reading frames

should be highly biased towards those

proteins that share the cellular location.

Cases where a protein is identified in

more than one location reveal possible

interactions that can be considered in

the context of the entire cell. Multi-

ple locations reflect functionally and

physically related subcellular regions,

including regions that undergo dynamic

interchange of proteins. The availabil-

ity of such proteomic libraries provides

the opportunity to probe how proteins

respond to external stimuli or growth

conditions over a selected period of

time. Complex protein interactions can

be mapped out by examining the effect

of the proteome localization due to

alterations of specific proteins.

Imaging in organisms

Many of the spectroscopic techniques described in the previous chapters,

such as MRI and fluorescence, are being used in clinical settings to visual-

ize metabolic processes in the human body. Another imaging technique,

positron emission tomography (PET), is being considered increasingly for

patient treatment in the examination of molecular processes and their

failure in disease. In this technique, a certain process is targeted and a suit-

able probe designed. The probes sensed in PET are drugs or analogs that

have been labeled with radioisotopes. The measurements are sensitive so

that very low doses can be applied to minimize unforeseen side effects.

As the name implies, this technique is tomography-based, which means

that the resulting information is measured in three dimensions so that it

can be mapped onto the body.

414 PART 3 UNDERSTANDING BIOLOGICAL SYSTEMS USING PHYSICAL CHEMISTRY

PCR product

(a)

Chromosome

Homologous

recombination

Fusion protein NH 2 Protein COOH

ORF ORF

GFP HIS3MX6

GFP

Figure 19.9Localization studies using GFP. (a) The
strategy for the library-based attachment of the GFP
fusion products to over 6000 open reading frames
of the S. cerevisiae genome. Inclusion of the His3MX
gene permitted selection in histidine-free media.
(b) Fluorescence microscopy of the expressed fusion
products shows localization in different cellular areas
for different proteins. Modified from Huh et al. (2003).