Aron Broom

Modular Evolution and the Origins of Symmetry: Reconstruction of a Three-Fold Symmetric Globular Protein

The high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules.

Energetics of oligomeric protein folding and association

In nature, proteins most often exist as complexes, with many of these consisting of identical subunits. Understanding of the energetics governing the folding and misfolding of such homooligomeric proteins is central to understanding their function and misfunction, in disease or biotechnology. Much progress has been made in defining the mechanisms and thermodynamics of homooligomeric protein folding. In this review, we outline models as well as calorimetric and spectroscopic methods for characterizing oligomer folding, and describe extensive results obtained for diverse proteins, ranging from dimers to octamers and higher order aggregates. To our knowledge, this area has not been reviewed comprehensively in years, and the collective progress is impressive. The results provide evolutionary insights into the development of subunit interfaces, mechanisms of oligomer folding, and contributions of oligomerization to protein stability, function and regulation. Thermodynamic analyses have also proven valuable for understanding protein misfolding and aggregation mechanisms, suggesting new therapeutic avenues. Successful recent designs of novel, functional proteins demonstrate increased understanding of oligomer folding. Further rigorous analyses using multiple experimental and computational approaches are still required, however, to achieve consistent and accurate prediction of oligomer folding energetics. Modeling the energetics remains challenging but is a promising avenue for future advances.

Kinetic Stability of the Streptavidin–Biotin Interaction Enhanced in the Gas Phase

Results of the first detailed study of the structure and kinetic stability of the model high-affinity protein-ligand interaction between biotin (B) and the homotetrameric protein complex streptavidin (S(4)) in the gas phase are described. Collision cross sections (Ω) measured for protonated gaseous ions of free and ligand-bound truncated (residues 13-139) wild-type (WT) streptavidin, i.e., S(4)(n+) and (S(4)+4B)(n+) at charge states n = 12-16, were found to be independent of charge state and in agreement (within 10%) with values estimated for crystal structures reported for S(4) and (S(4)+4B). These results suggest that significant structural changes do not occur upon transfer of the complexes from solution to the gas phase by electrospray ionization. Temperature-dependent rate constants were measured for the loss of B from the protonated (S(4)+4B)(n+) ions. Over the temperature range investigated, the kinetic stability increases with decreasing charge state, from n = 16 to 13, but is indistinguishable for n = 12 and 13. A comparison of the activation energies (E(a)) measured for the loss of B from the (S(4)+4B)(13+) ions composed of WT streptavidin and five binding site mutants (Trp79Phe, Trp108Phe, Trp120Phe, Ser27Ala, and Tyr43Ala) suggests that at least some of the specific intermolecular interactions are preserved in the gas phase. The results of molecular dynamics simulations performed on WT (S(4)+4B)(12+) ions with different charge configurations support this conclusion. The most significant finding of this study is that the gaseous WT (S(4)+4B)(n+) ions at n = 12-14, owing to a much larger E(a) (by as much as 13 kcal mol(-1)) for the loss of B, are dramatically more stable kinetically at 25 °C than the (S(4)+4B) complex in aqueous neutral solution. The differences in E(a) values measured for the gaseous (S(4)+4B)(n+) ions and solvated (S(4)+4B) complex can be largely accounted for by a late dissociative transition state and the rehydration of B and the protein binding cavity in solution.

Nonnative interactions regulate folding and switching of myristoylated protein

We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin- and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.

Exploring the relationships between protein sequence, structure and solubility

Aggregation can be thought of as a form of protein folding in which intermolecular associations lead to the formation of large, insoluble assemblies. Various types of aggregates can be differentiated by their internal structures and gross morphologies (e.g., fibrillar or amorphous), and the ability to accurately predict the likelihood of their formation by a given polypeptide is of great practical utility in the fields of biology (including the study of disease), biotechnology, and biomaterials research. Here we review aggregation/solubility prediction methods and selected applications thereof. The development of increasingly sophisticated methods that incorporate knowledge of conformations possibly adopted by aggregating polypeptide monomers and predict the internal structure of aggregates is improving the accuracy of the predictions and continually expanding the range of applications.

Folding and association of thermophilic dimeric and trimeric DsrEFH proteins: Tm0979 and Mth1491.

Although the majority of natural proteins exist as protein-protein complexes, the molecular basis for the formation and regulation of such interactions and the evolution of protein interfaces remain poorly understood. We have investigated these phenomena by characterizing the thermal and chemical denaturation of thermophilic DsrEFH proteins that have a common subunit fold but distinct quaternary structures: homodimeric Tm0979 and homotrimeric Mth1491. Tm0979 forms a moderate affinity dimer, and a monomeric intermediate is readily populated at equilibrium and during folding kinetics. In contrast, the Mth1491 trimer has extremely high stability, so that a monomeric form is not measurably populated at equilibrium, although it may be during folding kinetics. A common mechanism for evolution of quaternary structures may be facile formation of a relatively stable monomeric species, with stabilizing intermolecular interactions centering on alternative environments for a beta-strand at the edge of the monomer, augmented by malleable hydrophobic interactions. The exceptional trimer stability arises from a remarkably slow unfolding rate constant, 6.5 x 10(-13) s(-1), which is a common characteristic of highly stable thermophilic and/or oligomeric proteins. The folding characteristics of Tm0979 and Mth1491 have interesting implications for assembly and regulation of homo- and heterooligomeric proteins in vivo.

Designed protein reveals structural determinants of extreme kinetic stability

Significance Much research has focused on the molecular basis for protein thermodynamic stability; by comparison, kinetic stability is much less understood. Thermodynamics define the equilibrium fraction of unfolded protein while kinetics define the rate of unfolding; the latter can be of great practical importance for ensuring a protein remains folded under biological conditions. Using extensive experimental and modeling analyses we show that ThreeFoil, a small glycan binding protein without disulfides, exhibits outstanding kinetic stability against chemical denaturation and proteolytic degradation. We demonstrate that high kinetic stability is successfully modeled in terms of extensive long-range intramolecular interactions. These results show that topological complexity is a key determinant of kinetic stability which should help in designing proteins to withstand harsh conditions. The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge.

Protein unfolding rates correlate as strongly as folding rates with native structure

Although the folding rates of proteins have been studied extensively, both experimentally and theoretically, and many native state topological parameters have been proposed to correlate with or predict these rates, unfolding rates have received much less attention. Moreover, unfolding rates have generally been thought either to not relate to native topology in the same manner as folding rates, perhaps depending on different topological parameters, or to be more difficult to predict. Using a dataset of 108 proteins including two‐state and multistate folders, we find that both unfolding and folding rates correlate strongly, and comparably well, with well‐established measures of native topology, the absolute contact order and the long range order, with correlation coefficient values of 0.75 or higher. In addition, compared to folding rates, the absolute values of unfolding rates vary more strongly with native topology, have a larger range of values, and correlate better with thermodynamic stability. Similar trends are observed for subsets of different protein structural classes. Taken together, these results suggest that choosing a scaffold for protein engineering may require a compromise between a simple topology that will fold sufficiently quickly but also unfold quickly, and a complex topology that will unfold slowly and hence have kinetic stability, but fold slowly. These observations, together with the established role of kinetic stability in determining resistance to thermal and chemical denaturation as well as proteases, have important implications for understanding fundamental aspects of protein unfolding and folding and for protein engineering and design.

Computational tools help improve protein stability but with a solubility tradeoff

Accurately predicting changes in protein stability upon amino acid substitution is a much sought after goal. Destabilizing mutations are often implicated in disease, whereas stabilizing mutations are of great value for industrial and therapeutic biotechnology. Increasing protein stability is an especially challenging task, with random substitution yielding stabilizing mutations in only ∼2% of cases. To overcome this bottleneck, computational tools that aim to predict the effect of mutations have been developed; however, achieving accuracy and consistency remains challenging. Here, we combined 11 freely available tools into a meta-predictor (meieringlab.uwaterloo.ca/stabilitypredict/). Validation against ∼600 experimental mutations indicated that our meta-predictor has improved performance over any of the individual tools. The meta-predictor was then used to recommend 10 mutations in a previously designed protein of moderate thermodynamic stability, ThreeFoil. Experimental characterization showed that four mutations increased protein stability and could be amplified through ThreeFoils structural symmetry to yield several multiple mutants with >2-kcal/mol stabilization. By avoiding residues within functional ties, we could maintain ThreeFoils glycan-binding capacity. Despite successfully achieving substantial stabilization, however, almost all mutations decreased protein solubility, the most common cause of protein design failure. Examination of the 600-mutation data set revealed that stabilizing mutations on the protein surface tend to increase hydrophobicity and that the individual tools favor this approach to gain stability. Thus, whereas currently available tools can increase protein stability and combining them into a meta-predictor yields enhanced reliability, improvements to the potentials/force fields underlying these tools are needed to avoid gaining protein stability at the cost of solubility.

Using natural sequences and modularity to design common and novel protein topologies.

Protein design is still a challenging undertaking, often requiring multiple attempts or iterations for success. Typically, the source of failure is unclear, and scoring metrics appear similar between successful and failed cases. Nevertheless, the use of sequence statistics, modularity and symmetry from natural proteins, combined with computational design both at the coarse-grained and atomistic levels is propelling a new wave of design efforts to success. Here we highlight recent examples of design, showing how the wealth of natural protein sequence and topology data may be leveraged to reduce the search space and increase the likelihood of achieving desired outcomes.