What makes a protein a protein? Hydrophobic core designs that specify stability and structural properties

Abstract

Here we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four-helix-bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native-like properties of the wildtype, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with larger side chains causes a loss of native-like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native-like proteins.Keywords: hydrophobic core; molecular packing; molten globule; protein design What makes a protein a protein and distinguishes it from a collapsed polymer? A pressing goal of protein engineering and design is to understand the features that give rise to the physical properties associated with a stable, "native-like" protein structure. A well-developed secondary structure is essential and the intrinsic requirements for a-helix and P-sheet formation are becoming increasingly clear (Regan, 1994;Bryson et al., 1995). Although important for stability, the turn and loop connections between elements of secondary structure often do not play a principal role in specifying the structure and properties of a protein (Brunet et al., 1993;Castagnoli et al., 1994;Predki et al., 1996) Regan, in prep). By contrast, the packing of residues in the hydrophobic core appears to be extremely important for the structure, stability, and native-like properties of natural proteins (Kellis et al

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

“…8 When AI6 was crystallized, it was found in a parallel (or syn) topology, the opposite of that observed for wild-type Rop (anti, Fig. 7).…”

“…1). 8 We decided to initially mutate Cys38 and Cys52 to Ser/Ser, Ala/Ala, and Val/Val. The genes for these variants were produced by PCR assembly of four $62-mer synthetic oligonucleotides.…”

“…We performed differential scanning calorimetry on Ala/Val Rop and compared the values to wildtype from the work of Munson et al 8,19 (Table II). The T1 =2 values (temperature half-way through the calorimetric transition) at 0.3 mM are opposite those determined by CD spectroscopy at 50 lM (compare to Table I).…”

“…2,3 Remarkable successes in packing or repacking protein cores have come from rational and computational design, as well as directed evolution. [4][5][6][7][8] But there is still no accurate predictive model for the thermodynamic or kinetic consequences of even a single point mutation in the core of a protein.…”

“…17 Extensive mutations have been made to the core and loop of Rop to probe their effects on stability, folding and structure. 8,[18][19][20][21][22] Recently, we have reported in vivo and in vitro screens for the folding and stability of Rop, with the goal of correlating the effects of many kinds of mutations with changes in thermodynamics. 23,24 A proofof-principle library of mutations in the central core of Rop (I15 T19 L41 A45 to all amino acids with the NNK residue) produced a library of active variants with a range of physical properties (TJM and Lynne Regan, manuscript in preparation).…”