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.

Distinguishing specific and nonspecific interdomain interactions in multidomain proteins.

Multidomain proteins account for over two-thirds of the eukaryotic genome. Although there have been extensive studies into the biophysical properties of isolated domains, few have investigated how the domains interact. Spectrin is a well-characterized multidomain protein with domains linked in tandem array by contiguous helices. Several of these domains have been shown to be stabilized by their neighbors. Until now, this stabilization has been attributed to specific interactions between the natural neighbors, however we have recently observed that nonnatural neighboring domains can also induce a significant amount of stabilization. Here we investigate this nonnative stabilizing effect. We created spectrin-titin domain pairs of both spectrin R16 and R17 with a single titin I27 domain at either the N- or the C-terminus and found that spectrin domains are significantly stabilized, through slowed unfolding, by nonnative interactions at the C-terminus only. Of particular importance, we show that specific interactions between natural folded neighbors at either terminus confer even greater stability by additionally increasing the folding rate constants. We demonstrate that it is possible to distinguish between natural stabilizing interactions and nonspecific stabilizing effects through examination of the kinetics of well chosen mutant proteins. This work adds to the complexity of studying multidomain proteins.

Designing an extracellular matrix protein with enhanced mechanical stability.

The extracellular matrix proteins tenascin and fibronectin experience significant mechanical forces in vivo. Both contain a number of tandem repeating homologous fibronectin type III (fnIII) domains, and atomic force microscopy experiments have demonstrated that the mechanical strength of these domains can vary significantly. Previous work has shown that mutations in the core of an fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that domain significantly: The composition of the core is apparently crucial to the mechanical stability of these proteins. Based on these results, we have used rational redesign to increase the mechanical stability of the 10th fnIII domain of human fibronectin, FNfn10, which is directly involved in integrin binding. The hydrophobic core of FNfn10 was replaced with that of the homologous, mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc retains both the three-dimensional structure and the cell adhesion activity of FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of the engineered protein FNoTNc increase by approximately 20% to match those of TNfn3. Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions.

Spectrin domains lose cooperativity in forced unfolding.

Spectrin is a multidomain cytoskeletal protein, the component three-helix bundle domains are expected to experience mechanical force in vivo. In thermodynamic and kinetic studies, neighboring domains of chicken brain alpha-spectrin R16 and R17 have been shown to behave cooperatively. Is this cooperativity maintained under force? The effect of force on these spectrin domains was investigated using atomic force microscopy. The response of the individual domains to force was compared to that of the tandem repeat R1617. Importantly, nonhelical linkers (all-beta immunoglobulin domains) were used to avoid formation of nonnative helical linkers. We show that, in contrast to previous studies on spectrin repeats, only 3% of R1617 unfolding events gave an increase in contour length consistent with cooperative two-domain unfolding events. Furthermore, the unfolding forces for R1617 were the same as those for the unfolding of R16 or R17 alone. This is a strong indication that the cooperative unfolding behavior observed in the stopped-flow studies is absent between these spectrin domains when force is acting as a denaturant. Our evidence suggests that the rare double unfolding events result from misfolding between adjacent repeats. We suggest that this switch from cooperative to independent behavior allows multidomain proteins to maintain integrity under applied force.

Single molecule studies of protein folding using atomic force microscopy.

Atomic force microscopy (AFM) offers new insights into the ability of proteins to resist mechanical force. The technique has been opened up by the availability of easy-to-use instruments that are commercially available, so that the technique no longer relies on the need to build instruments in the lab. Indeed it may become common for AFM instruments to sit beside stopped-flow apparatus in protein folding laboratories. In this chapter, we describe the instrument set-up, the preparation of suitable protein substrate, and the collection of data. Data selection and analysis are more complex than for conventional stopped-flow ensemble studies, but offer new insights into the function of proteins in vivo.

Using model proteins to quantify the effects of pathogenic mutations in Ig-like proteins.

It has proved impossible to purify some proteins implicated in disease in sufficient quantities to allow a biophysical characterization of the effect of pathogenic mutations. To overcome this problem we have analyzed 37 different disease-causing mutations located in the L1 and IL2Rgamma proteins in well characterized related model proteins in which mutations that are identical or equivalent to pathogenic mutations were introduced. We show that data from these models are consistent and that changes in stability observed can be correlated to severity of disease, to correct trafficking within the cell and to in vitro ligand binding studies. Interestingly, we find that any mutations that cause a loss of stability of more than 2 kcal/mol are severely debilitating, even though some model proteins with these mutations can be easily expressed and analyzed. Furthermore we show that the severity of mutation can be predicted by a DeltaDeltaG(evolution) scale, a measure of conservation. Our results demonstrate that model proteins can be used to analyze disease-causing mutations when wild-type proteins are not stable enough to carry mutations for biophysical analysis.

The folding pathway of spectrin R17 from experiment and simulation: using experimentally validated MD simulations to characterize States hinted at by experiment.

We present an experimental and computational analysis of the folding pathway of the 17th domain of chicken brain alpha-spectrin, R17. Wild-type R17 folds in a two-state manner and the chevron plot (plot of the logarithm of the observed rate constant against concentration of urea) shows essentially linear folding and unfolding arms. A number of mutant proteins, however, show a change in slope of the unfolding arm at high concentration of denaturant, hinting at complexity in the folding landscape. Through a combination of mutational studies and high temperature molecular dynamics simulations we show that the folding of R17 can be described by a model with two sequential transition states separated by an intermediate species. The rate limiting transition state for folding in water has been characterized both through experimental Phi-value analysis and by simulation. In contrast, a detailed analysis of the transition state predicted to dominate under highly denaturing conditions is only possible by simulation.

Cooperative folding in a multi-domain protein.

Most protein domains are found in multi-domain proteins, yet most studies of protein folding have concentrated on small, single-domain proteins or on isolated domains from larger proteins. Spectrin domains are small (106 amino acid residues), independently folding domains consisting of three long alpha-helices. They are found in multi-domain proteins with a number of spectrin domains in tandem array. Structural studies have shown that in these arrays the last helix of one domain forms a continuous helix with the first helix of the following domain. It has been demonstrated that a number of spectrin domains are stabilised by their neighbours. Here we investigate the molecular basis for cooperativity between adjacent spectrin domains 16 and 17 from chicken brain alpha-spectrin (R16 and R17). We show that whereas the proteins unfold as a single cooperative unit at 25 degrees C, cooperativity is lost at higher temperatures and in the presence of stabilising salts. Mutations in the linker region also cause the cooperativity to be lost. However, the cooperativity does not rely on specific interactions in the linker region alone. Most mutations in the R17 domain cause a decrease in cooperativity, whereas proteins with mutations in the R16 domain still fold cooperatively. We propose a mechanism for this behaviour.

The folding of spectrin domains II: phi-value analysis of R16.

Studies on the folding of helical proteins have shown a wide range of different mechanisms and highlighted the importance of helical propensity as a factor in determining folding mechanism. Here, we contribute to this interesting field with the protein engineering phi-value analysis of the 16th domain of chicken brain alpha-spectrin, R16. The fortuitous curvature seen in the unfolding arm of the chevron plot allows us to investigate both early and late events in folding. R16 is the first two-state helical protein for which this has been possible.

Parallel protein-unfolding pathways revealed and mapped.

Theoretical studies of protein folding suggest that multiple folding pathways should exist, but there is little experimental evidence to support this. Here we demonstrate changes in the flux between different transition states on parallel folding pathways, resulting in unprecedented upward curvature in the denaturant-dependent unfolding kinetics of a beta-sandwich protein. As denaturant concentration increases, the highly compact transition state of one pathway becomes destabilized and the dominant flux of protein molecules shifts toward another pathway with a less structured transition state. Furthermore, point mutations alter the relative accessibility of the pathways, allowing the structure of two transition states on separate, direct folding pathways to be mapped by systematic Phi-value analysis. It has been suggested that pathways with diffuse rather than localized transition states are evolutionarily selected to prevent misfolding, and indeed we find that the transition state favored at high concentrations of denaturant is more polarized than the physiologically relevant one.