Folding pathway of an Ig domain is conserved on and off the ribosome.

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-ß Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.

Cotranslational folding of spectrin domains via partially structured states.

How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.

Smoothing a rugged protein folding landscape by sequence-based redesign.

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.

Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain.

Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100°C, a ΔG(D-N) of 15.5 kcal mol(-1) and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 Å and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.

A mechanistic model for amorphous protein aggregation of immunoglobulin-like domains.

Protein aggregation is associated with many debilitating diseases including Alzheimer's, Parkinson's, and light-chain amyloidosis (AL). Additionally, such aggregation is a major problem in an industrial setting where antibody therapeutics often require high local concentrations of protein domains to be stable for substantial periods of time. However, despite a plethora of research in this field, dating back over 50 years, there is still no consensus on the mechanistic basis for protein aggregation. Here we use experimental data to derive a mechanistic model that well describes the aggregation of Titin I27, an immunoglobulin-like domain. Importantly, we find that models that are suitable for nucleated fibril formation do not fit our aggregation data. Instead, we show that aggregation proceeds via the addition of activated dimers, and that the rate of aggregation is dependent on the surface area of the aggregate. Moreover, we suggest that the "lag time" seen in these studies is not the time needed for a nucleation event to occur, but rather it is the time taken for the concentration of activated dimers to cross a particular solubility limit. These findings are reminiscent of the Finke-Watzky aggregation mechanism, originally based on nanocluster formation and suggest that amorphous aggregation processes may require mechanistic schemes that are substantially different from those of linear fibril formation.

Take home lessons from studies of related proteins.

The 'Fold Approach' involves a detailed analysis of the folding of several topologically, structurally and/or evolutionarily related proteins. Such studies can reveal determinants of the folding mechanism beyond the gross topology, and can dissect the residues required for folding from those required for stability or function. While this approach has not yet matured to the point where we can predict the native conformation of any polypeptide chain in silico, it has been able to highlight, amongst others, the specific residues that are responsible for nucleation, pathway malleability, kinetic intermediates, chain knotting, internal friction and Paracelsus switches. Some of the most interesting discoveries have resulted from the attempt to explain differences between homologues.

Understanding pathogenic single-nucleotide polymorphisms in multidomain proteins--studies of isolated domains are not enough.

Studying the effects of pathogenic mutations is more complex in multidomain proteins when compared with single domains: mutations occurring at domain boundaries may have a large effect on a neighbouring domain that will not be detected in a single-domain system. To demonstrate this, we present a study that utilizes well-characterized model protein domains from human spectrin to investigate the effect of disease- and non-disease-causing single point mutations occurring at the boundaries of human spectrin repeats. Our results show that mutations in the single domains have no clear correlation with stability and disease; however, when studied in a tandem model system, the disease-causing mutations are shown to disrupt stabilizing interactions that exist between domains. This results in a much larger decrease in stability than would otherwise have been predicted, and demonstrates the importance of studying such mutations in the correct protein context.

The morphology of decorated amyloid fibers is controlled by the conformation and position of the displayed protein.

Self-assembled structures capable of mediating electron transfer are an attractive scientific and technological goal. Therefore, systematic variants of SH3-Cytochrome b(562) fusion proteins were designed to make amyloid fibers displaying heme-b(562) electron transfer complexes. TEM and AFM data show that fiber morphology responds systematically to placement of b(562) within the fusion proteins. UV-vis spectroscopy shows that, for the fusion proteins under test, only half the fiber-borne b(562) binds heme with high affinity. Cofactor binding also improves the AFM imaging properties and changes the fiber morphology through changes in cytochrome conformation. Systematic observations and measurements of fiber geometry suggest that longitudinal registry of subfilaments within the fiber, mediated by the interaction and conformation of the displayed proteins and their interaction with surfaces, gives rise to the observed morphologies, including defects and kinks. Of most interest is the role of small molecule modulation of fiber structure and mechanical stability. A minimum complexity model is proposed to capture and explain the fiber morphology in the light of these results. Understanding the complex interplay between these factors will enable a fiber design that supports longitudinal electron transfer.

Affinity maturation of phage display antibody populations using ribosome display.

Ribsosome display is a PCR-based in vitro display technology that it well suited for the selection and evolution of high-affinity antibodies. In particular, ribosome display lends itself to the evolution of functional characteristics, such as potency, and thereby facilitates the production of therapeutic antibodies from lead candidates. In this chapter, we describe how to mature large phage display antibody populations (>10(7)) by performing increasingly stringent selections with decreasing antigen concentration. This process takes advantage of ribosome display's intrinsic ability to evolve sequence during selection. Ribosome display can also be used as a complementary tool to phage display for isolating high-affinity antibodies from naïve libraries. Ultimately, maturation of large antibody populations by ribosome display will help to speed up the process of generating antibody therapeutics.

What lessons can be learned from studying the folding of homologous proteins?

The studies of the folding of structurally related proteins have proved to be a very important tool for investigating protein folding. Here we review some of the insights that have been gained from such studies. Our highlighted studies show just how such an investigation should be designed and emphasise the importance of the synergy between experiment and theory. We also stress the importance of choosing the right system carefully, exploiting the excellent structural and sequence databases at our disposal.

Folding of a LysM domain: entropy-enthalpy compensation in the transition state of an ideal two-state folder.

Protein-engineering methods (Phi-values) were used to investigate the folding transition state of a lysin motif (LysM) domain from Escherichia coli membrane-bound lytic murein transglycosylase D. This domain consists of just 48 structured residues in a symmetrical betaalphaalphabeta arrangement and is the smallest alphabeta protein yet investigated using these methods. An extensive mutational analysis revealed a highly robust folding pathway with no detectable transition state plasticity, indicating that LysM is an example of an ideal two-state folder. The pattern of Phi-values denotes a highly polarised transition state, with significant formation of the helices but no structure within the beta-sheet. Remarkably, this transition state remains polarised after circularisation of the domain, and exhibits an identical Phi-value pattern; however, the interactions within the transition state are uniformly weaker in the circular variant. This observation is supported by results from an Eyring analysis of the folding rates of the two proteins. We propose that the folding pathway of LysM is dominated by enthalpic rather than entropic considerations, and suggest that the lower entropy cost of formation of the circular transition state is balanced, to some extent, by the lower enthalpy of contacts within this structure.

Studying the folding of multidomain proteins.

There have been relatively few detailed comprehensive studies of the folding of protein domains (or modules) in the context of their natural covalently linked neighbors. This is despite the fact that a significant proportion of the proteome consists of multidomain proteins. In this review we highlight some key experimental investigations of the folding of multidomain proteins to draw attention to the difficulties that can arise in analyzing such systems. The evidence suggests that interdomain interactions can significantly affect stability, folding, and unfolding rates. However, preliminary studies suggest that folding pathways are unaffected-to this extent domains can be truly considered to be independent folding units. Nonetheless, it is clear that interactions between domains cannot be ignored, in particular when considering the effects of mutations.

The folding and evolution of multidomain proteins.

Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent. Furthermore, recent studies indicate that multidomain proteins have evolved mechanisms to minimize the problems of interdomain misfolding.

BPPred: a Web-based computational tool for predicting biophysical parameters of proteins.

We exploit the availability of recent experimental data on a variety of proteins to develop a Web-based prediction algorithm (BPPred) to calculate several biophysical parameters commonly used to describe the folding process. These parameters include the equilibrium m-values, the length of proteins, and the changes upon unfolding in the solvent-accessible surface area, in the heat capacity, and in the radius of gyration. We also show that the knowledge of any one of these quantities allows an estimate of the others to be obtained, and describe the confidence limits with which these estimations can be made. Furthermore, we discuss how the kinetic m-values, or the Beta Tanford values, may provide an estimate of the solvent-accessible surface area and the radius of gyration of the transition state for protein folding. Taken together, these results suggest that BPPred should represent a valuable tool for interpreting experimental measurements, as well as the results of molecular dynamics simulations.