De novo design of the hydrophobic core of ubiquitin

Abstract

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that P-sheet structures have more stringent packing requirements than a-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold.Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.Keywords: computational; genetic algorithm; hydrophobic core packing; protein design; ubiquitin Protein design is an extremely powerful method for critically testing the relationship between the primary sequence and threedimensional structure of proteins. One goal of protein design is to reconstruct known three-dimensional folds from completely novel amino acid sequences. The concepts used to design the amino acid sequences constitute a hypothesis of what interactions are necessary for a protein to adopt a given three-dimensional structure with the properties of natural proteins, and at what level of detail these interactions must be considered.The hypotheses of early protein design efforts were based on the assumption that simple empirical rules would be sufficient. These designs included, foremost, patterns of hydrophobic and hydrophilic residues, and to a lesser extent, secondary structure propensities and poten...

Side-chain and backbone flexibility in protein core design 1 1Edited by A. R. Fersht

“…A more common approach includes additional discrete rotamers at de®ned increments, for example at AE 15 or AE one standard deviation from the statistically derived rotamer value (Dahiyat & Mayo, 1996;Tuffery et al, 1991). For our analysis, we have also used a version of our ®xed backbone algorithm that incorporates a wide range of¯exibility into the side-chains, with ®ne sampling of side-chain dihedral angles (5 ) at almost all possible angles (Lazar et al, 1997). An analysis of side-chain¯exibility effects is crucial at this stage, since the¯exible backbone method allows complete side-chain¯exibility as well.…”

“…The ROC programs used in this report have been described (Desjarlais & Handel, 1995;Lazar et al, 1997). Brie¯y, a ®xed backbone structure and a list of core residues is read into the program, and side-chain identities and/or conformers for each core position are sampled from a rotamer library.…”

“…A small number of these methods have now been con®rmed by experimental characterization of core variants designed with the programs. In our own previous work, we demonstrated the successful design of several hydrophobic core variants of the proteins 434 cro and ubiquitin using a genetic algorithm called ROC (repacking of cores) which assumes a ®xed backbone structure (Desjarlais & Handel, 1995;Lazar et al, 1997;Johnson et al, 1999). Mayo and co-workers have demonstrated similar success in the design of alternative core sequences for the proteins GCN4 and the B1 domain of protein G (Dahiyat & Mayo, 1996.…”

“…Almost all of these methods involve the use of a ®xed backbone structure. Much of this work has focused on evaluation of the thermodynamic consequences of core mutations (Dahiyat & Mayo, 1996;Desjarlais & Handel, 1995;Hellinga & Richards, 1994;Lazar et al, 1997;Lee & Levitt, 1991), prediction of hydrophobic core sequences (Dahiyat & Mayo, 1996;Desjarlais & Handel, 1995;Kono & Doi, 1994;Lazar et al, 1997;Ponder & Richards, 1987), or prediction of core structure (Dahiyat & Mayo, 1996;Desjarlais & Handel, 1995;Harbury et al, 1995;Hellinga & Richards, 1994;Kono & Doi, 1994;Lee & Subbiah, 1991;Tuffery et al, 1991;Wilson et al, 1993). A small number of these methods have now been con®rmed by experimental characterization of core variants designed with the programs.…”

Stabilizing the subtilisin BPN' pro‐domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?

“…Therefore, in the context of all other loci, T4 lysozyme can tolerate a loss in stability of 7 kcal/mol as a result of the 10 methionine substitutions in its core and maintain its native structure. Other studies have shown that computer-designed hydrophobic cores of equal volume are not equal in stability but rather result in a range of stabilities (Desjarlais & Handel, 1995;Lazar et al, 1997).…”