Hydrophobic core malleability of a de novo designed three-helix bundle protein.
De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of alpha(3)D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing ( approximately 1.5 kcal mol(-1)) to the tertiary structure of alpha(3)D. The value of DeltaC(p) for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-alpha(3)D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha(3)D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that alpha(3)D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins.
Walsh
S T
ST
The Johnson Research Foundation, University of Pennsylvania, Philadelphia, PA, 19104-6059, USA.
Sukharev
V I
VI
Betz
S F
SF
Vekshin
N L
NL
DeGrado
W F
WF
D019943:Amino Acid Substitution / Q000235:genetics; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D018583:Pliability; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012997:Solvents; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation
D011506:Proteins; D012997:Solvents; D019791:Guanidine