Cysteine-free Rop: a four-helix bundle core mutant has wild-type stability and structure but dramatically different unfolding kinetics

Abstract

Cysteine residues can complicate the folding and storage of proteins due to improper formation of disulfide bonds or oxidation of residues that are natively reduced. Wild-type Rop is a homodimeric four-helix bundle protein and an important model for protein design in the understanding of protein stability, structure and folding kinetics. In the native state, Rop has two buried, reduced cysteine residues in its core, but these are prone to oxidation in destabilized variants, particularly upon extended storage. To circumvent this problem, we designed and characterized a Cys-free variant of Rop, including solving the 2.3 A X-ray crystal structure. We show that the C38A C52V variant has similar structure, stability and in vivo activity to wild-type Rop, but that it has dramatically faster unfolding kinetics like virtually every other mutant of Rop that has been characterized. This cysteine-free Rop has already proven useful for studies on solution topology and on the relationship of core mutations to stability. It also suggests a general strategy for removal of pairs of Cys residues in proteins, both to make them more experimentally tractable and to improve their storage properties for therapeutic or industrial purposes.

Figure 1

The structure of wild-type Rop. Rop is an antiparallel homodimer of 63 amino acid monomers, placing the reduced Cys38 and Cy52 residues near each other across the dimer interface. At right, in the monomer, residues in the hydrophobic core of Rop are shown as spheres and labeled with their position (a, d, or e) in the heptad repeat. Note that in the last repeat, R55 (d) is mostly exposed and F56 (e) instead packs into the core. Rendered from 1ROP with PyMOL (Delano Scientific).

Figure 2

Packing and interactions of Cys38 and Cys52 in wild-type Rop. Residues within 5 Å are shown at the top, and atoms within 5 Å of the Cys sulfur atoms are shown below. Mint-colored carbons are from the other monomer. At left, Cys38 may make a hydrogen bond to the carbonyl oxygen of Thr19, but it is well-packed with mostly hydrophobic residues nearby. At right, Cys52 is slightly further from the carbonyl oxygen of Leu48 but appears to be much less tightly packed. Rendered from 1ROP with PyMOL.

Figure 3

In vivo activity of Rop cysteine variants. The Ser/Ser variant is inactive, with low fluorescence like the negative control. The Ala/Ala, Val/Val, and Ala/Val variants are as active as wild-type Rop.

Figure 4

Thermal and urea denaturation of Rop cysteine variants observed by CD spectroscopy. The Ala/Val variant is slightly more thermally stable than wild-type Rop, while Ala/Ala is much less stable. The Ala/Val variant is slightly destabilized to urea denaturation compared to wild-type, but it is much more stable than Ala/Ala.

Figure 5

Unfolding kinetics of wild-type and Ala/Val Rop from urea observed by CD spectroscopy. Each log k_u (where k is in s⁻¹) is plotted as a function of urea concentration. Ala/Val Rop's unfolding rate constant is about 140-fold higher than wild-type in zero denaturant.

Figure 6

X-ray crystal structure of Ala/Val Rop. (a) Overlay of the backbones of the symmetry-derived dimers of wild-type Rop (cyan carbons) and Ala/Val Rop (green carbons). The positions of the C38A and C52V mutations are shown as 50% transparent spheres for the wild-type Cys side chains. There is little change in the overall structure. (b) Overlay of the monomer backbones with core residues rendered as thick sticks. The backbone in helix 1 opposite C38A has moved slightly toward position 38. Near C52V, a small movement of the backbone has displaced F56 toward the open end of the hairpin. (c) Resides with atoms within 5 Å of the Cys38 sulfur are nearly unchanged except for the slight backbone shift toward the site of mutation. (d) It is evident that Val38 would have clashed with position of Phe56 in the wild-type, and it is displaced slightly by movement of the backbone. In (c) and (d), C38, C52, and F56 are shown in 50% transparent spheres with cyan carbons in the wild-type, and A38, V52, and F56 are shown in solid spheres with green carbons in Ala/Val. Rendered with PyMOL from 1ROP and the structure solved here, 3K79.

Figure 7

Topologies of Rop variants. Wild-type Rop (1ROP, anti) and AI6 Rop (1F4M, syn) were rendered with PyMOL. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]