Here we obtain the data needed to predict chemical interactions of polyethylene glycols (PEGs) and glycerol with proteins and related organic compounds, and thereby interpret or predict chemical effects of PEGs on protein processes. To accomplish this we determine interactions of glycerol and tetraEG with >30 model compounds displaying the major C, N, and O functional groups of proteins. Analysis of these data yields coefficients (α-values) quantifying interactions of glycerol, tetraEG and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with aromatic C, amide N, and cationic N, but unfavorably with amide O, carboxylate O and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliphatic C are quite favorable, while interactions of glycerol and PEG end groups with aliphatic C are not. Hence tetraEG and PEG 300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD) while glycerol, di- and mono-ethylene glycol are stabilizers. Favorable interactions with aromatic and aliphatic C explain why PEG400 greatly increases the solubility of aromatic hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α-values reported here, chemical contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA), and separated from excluded volume effects.

Consensus sequence design offers a promising strategy for designing proteins of high stability while retaining biological activity since it draws upon an evolutionary history in which residues important for both stability and function are likely to be conserved. Although there have been several reports of successful consensus design of individual targets, it is unclear from these anecdotal studies how often this approach succeeds and how often it fails. Here, we attempt to assess generality by designing consensus sequences for a set of six protein families with a range of chain lengths, structures, and activities. We characterize the resulting consensus proteins for stability, structure, and biological activities in an unbiased way. We find that all six consensus proteins adopt cooperatively folded structures in solution. Strikingly, four of six of these consensus proteins show increased thermodynamic stability over naturally occurring homologs. Each consensus protein tested for function maintained at least partial biological activity. Although peptide binding affinity by a consensus-designed SH3 is rather low, K m values for consensus enzymes are similar to values from extant homologs. Although consensus enzymes are slower than extant homologs at low temperature, they are faster than some thermophilic enzymes at high temperature. An analysis of sequence properties shows consensus proteins to be enriched in charged residues, and rarified in uncharged polar residues. Sequence differences between consensus and extant homologs are predominantly located at weakly conserved surface residues, highlighting the importance of these residues in the success of the consensus strategy.protein stability | protein design | consensus sequence

There is considerable interest in generating proteins with both high stability and high activity for biomedical and industrial purposes. One approach that has been used successfully to increase the stability of linear repeat proteins is consensus design. It is unclear the extent over which the consensus design approach can be used to produce folded and hyperstable proteins, and importantly, whether such stabilized proteins would retain function. Here we extend the consensus strategy to design a globular protein. We show that a consensus-designed homeodomain (HD) sequence adopts a cooperatively folded homeodomain structure. The unfolding free energy of the consensus-HD is 5 kcal·mol−1 higher than that of the naturally-occurring engrailed-HD from Drosophila melanogaster. Remarkably, the consensus-HD binds the engrailed-HD cognate DNA in a similar mode as the engrailed-HD with approximately 100-fold higher affinity. 15N relaxation studies show a decrease in psec-nsec backbone dynamics in the free state of consensus-HD, suggesting that increased affinity is not a result of increased plasticity. In addition to demonstrating the potential for consensus design of globular proteins with increased stability, these results demonstrate that greatly stabilized proteins can bind cognate substrates with increased affinities, showing that high stability is compatible with function.

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A major goal of protein design is to create proteins that have high stability and biological activity. Drawing on evolutionary information encoded within extant protein sequences, consensus sequence design has produced several successes in achieving this goal. Here we explore the generality with which consensus design can be used to enhance protein stability and maintain biological activity. By designing and characterizing consensus sequences for six unrelated protein families, we find that consensus design shows high success rates in creating well-folded, hyperstable proteins that retain biological activities. Remarkably, many of these consensus proteins show higher stabilities than naturally-occurring sequences of their respective protein families. Our study highlights the utility of consensus sequence design and informs the mechanisms by which it works.