Probing the role of packing specificity in protein design

Abstract

By using a protein-design algorithm that quantitatively considers side-chain packing, the effect of specific steric constraints on protein design was assessed in the core of the streptococcal protein G beta1 domain. The strength of packing constraints used in the design was varied, resulting in core sequences that reflected differing amounts of packing specificity. The structural flexibility and stability of several of the designed proteins were experimentally determined and showed a trend from well-ordered to highly mobile structures as the degree of packing specificity in the design decreased. This trend both demonstrates that the inclusion of specific packing interactions is necessary for the design of native-like proteins and defines a useful range of packing specificity for the design algorithm. In addition, an analysis of the modeled protein structures suggested that penalizing for exposed hydrophobic surface area can improve design performance.

Figure 1

Secondary structure and thermal stability of α90, α85, α70, and α107. (A) Far-UV CD spectra. (B) Thermal denaturation monitored by CD.

Figure 2

Proton NMR spectra of α90, α85, α70, α107, and α85W43V. The decrease in dispersion from α90 to α85 to α70 reflects a graded decrease in protein structural order. α107 appears unfolded. α85W43V has narrower lines and greater dispersion than α85, indicating that the single Trp → Val mutation reduced conformational flexibility. The sharp peaks at 8.45 and 0.15 ppm in the α70 spectrum are impurities.

Figure 3

Amide hydrogen–deuterium exchange kinetics of α90, α85, α70, α107, and α85W43V. Total area of exchangeable peaks, expressed as number of protons, as a function of exchange time at 25°C and pH 5.5.

Figure 4

Core packing arrangements predicted by DEE for α90 (Upper) and α85 (Lower). Only side chains for residues 34, 39, 43, 52, and 54 are shown. In α90, Trp-43 buries more than 90% of its surface area. In α85, Trp-43 is only 46% buried and is rotated into solvent to avoid steric clashes with Leu-34 and Phe-52, which occupy a larger volume than Ala-34 and Val-52 in α90. Figures were produced with molmol (33).