De novo design of the hydrophobic cores of proteins.
We have developed and experimentally tested a novel computational approach for the de novo design of hydrophobic cores. A pair of computer programs has been written, the first of which creates a "custom" rotamer library for potential hydrophobic residues, based on the backbone structure of the protein of interest. The second program uses a genetic algorithm to globally optimize for a low energy core sequence and structure, using the custom rotamer library as input. Success of the programs in predicting the sequences of native proteins indicates that they should be effective tools for protein design. Using these programs, we have designed and engineered several variants of the phage 434 cro protein, containing five, seven, or eight sequence changes in the hydrophobic core. As controls, we have produced a variant consisting of a randomly generated core with six sequence changes but equal volume relative to the native core and a variant with a "minimalist" core containing predominantly leucine residues. Two of the designs, including one with eight core sequence changes, have thermal stabilities comparable to the native protein, whereas the third design and the minimalist protein are significantly destabilized. The randomly designed control is completely unfolded under equivalent conditions. These results suggest that rational de novo design of hydrophobic cores is feasible, and stress the importance of specific packing interactions for the stability of proteins. A surprising aspect of the results is that all of the variants display highly cooperative thermal denaturation curves and reasonably dispersed NMR spectra. This suggests that the non-core residues of a protein play a significant role in determining the uniqueness of the folded structure.
Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA.
J R
JR
Desjarlais
T M
TM
Handel
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001435:Bacteriophages; D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry; D015847:Interleukin-4 / Q000737:chemistry; D007930:Leucine; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins; D012097:Repressor Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012984:Software; D013879:Thioredoxins / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D016222:Fibroblast Growth Factor 2; D015847:Interleukin-4; D013879:Thioredoxins; D009113:Muramidase; D007930:Leucine