Chapter 7 – Complementary Experimental Tools  267

Normally, however, cysteine residues would be buried deep in the inaccessible hydrophobic

core of a protein often present in the form of two nearby cysteine molecules bound together

via their respective sulfur atoms to form a disulfide bridge –​S–​S–​ (the subsequent cysteine

dimer is called cystine), which stabilize a folded protein structure. Chemically interfering

with the sulfhydryl group’s native cysteine amino acid residues can, therefore, change the

structure and function of the protein.

However, there are many proteins that contain no native cysteine residues. This is possibly

due to the function of these proteins requiring significant dynamic molecular conformational

changes that may be inhibited by the presence of –​S–​S–​ bonds in the structure. For these, it

is possible to introduce one or more foreign cysteine residues by modification of the DNA

encoding the protein using genetic engineering at specific sequence DNA locations. This

technique is an example of site-​directed mutagenesis (SDM), here specifically site-​directed

cysteine mutagenesis discussed later in this chapter. By introducing nonnative cysteines in

this way, they can be free to be used for chemical conjugation reactions while minimizing

impairment to the protein’s original biological function (though note that, in practice, signifi­

cant optimization is often still involved in finding the best candidate locations in a protein

sequence for a nonnative cysteine residue so as not to affect its biological function).

Binding to cysteine residues is also the most common method used in attaching spin labels

for ESR (see Chapter 5), especially through the cross-​linker chemical methanethiosulfonate

that contains an –​NO group with a strong ESR signal response.

7.2.3  ANTIBODIES

An antibody, or immunoglobulin (Ig), is a complex protein with bound sugar groups

produced by cells of the immune system in animals to bind to specific harmful infecting

agents in the body, such as bacteria and viruses. The basic structure of the most common

class of antibody is Y-​shaped (Figure 7.1), with a high molecular weight of ~150 kDa. The

types (or isotypes) of antibodies of this class are mostly found in mammals and are IgD (found

in milk/​saliva), IgE (commonly produced in allergic responses), and IgG, which are produced

in several immune responses and are the most widely used in biophysical techniques. Larger

variants consisting of multiple Y-​shaped subunits include IgA (a Y-​subunit dimer) and IgM (a

Y-​subunit pentamer). Other antibodies include IgW (found in sharks and skates, structurally

similar to IgD) and IgY (found in birds and reptiles).

FIGURE 7.1  Antibody labeling. Use of (a) immunoglobulin IgG antibody directly and (b) IgG

as a primary and a secondary IgG antibody, which is labeled with a biophysical tag that binds to

the Fc region. (c) Fab fragments can also be used directly.