Madeleine B. Borgia

Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins.

A large range of debilitating medical conditions is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains. Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding. Here we use single-molecule Förster resonance energy transfer to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.

Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy

Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes.

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments.

Significance In the interior of a cell, the volume accessible to each protein molecule is restricted by the presence of the large number of other macromolecules. Such a crowded environment is known to affect the stability and folding rates of proteins. In the case of intrinsically disordered proteins (IDPs), however, a class of proteins that lack stable structure, much less is known about the role of crowding effects. We have quantified the conformational changes occurring in IDPs in the presence of high concentrations of different polymers that act as crowding agents. Using single-molecule spectroscopy, we have identified effects that are typical of polymer solutions and have direct implications for the behavior of IDPs within the cell. Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concentration, but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the observed behavior can be explained quantitatively if the polymeric nature of both the IDPs and the crowding molecules is taken into account explicitly. Our results suggest that excluded volume interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu.

Temperature-dependent solvation modulates the dimensions of disordered proteins

Significance A large number of naturally occurring proteins are now known to be unstructured under physiological conditions. Many of these intrinsically disordered proteins (IDPs) bind other biological macromolecules or ligands and are involved in important regulatory processes in the cell. For understanding the structural basis of these functional properties, it is essential to quantify the balance of interactions that modulate the heterogeneous conformational distributions of IDPs and unfolded proteins in general. In contrast to the behavior expected for simple polymers with temperature-independent intramolecular interactions, unfolded proteins become more compact when the temperature is raised. Here, we show that the temperature dependence of the interactions of the constituent amino acid residues with the aqueous solvent have a dominant effect on this behavior. For disordered proteins, the dimensions of the chain are an important property that is sensitive to environmental conditions. We have used single-molecule Förster resonance energy transfer to probe the temperature-induced chain collapse of five unfolded or intrinsically disordered proteins. Because this behavior is sensitive to the details of intrachain and chain–solvent interactions, the collapse allows us to probe the physical interactions governing the dimensions of disordered proteins. We find that each of the proteins undergoes a collapse with increasing temperature, with the most hydrophobic one, λ-repressor, undergoing a reexpansion at the highest temperatures. Although such a collapse might be expected due to the temperature dependence of the classical “hydrophobic effect,” remarkably we find that the largest collapse occurs for the most hydrophilic, charged sequences. Using a combination of theory and simulation, we show that this result can be rationalized in terms of the temperature-dependent solvation free energies of the constituent amino acids, with the solvation properties of the most hydrophilic residues playing a large part in determining the collapse.

Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods.

There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no such increase of the radius of gyration (Rg). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of single-molecule Förster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.

Transient misfolding dominates multidomain protein folding

Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

Extreme disorder in an ultrahigh-affinity protein complex

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

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.

Empirical Optimization of Interactions between Proteins and Chemical Denaturants in Molecular Simulations

Chemical denaturants are the most commonly used perturbation applied to study protein stability and folding kinetics as well as the properties of unfolded polypeptides. We build on recent work balancing the interactions of proteins and water, and accurate models for the solution properties of urea and guanidinium chloride, to develop a combined force field that is able to capture the strength of interactions between proteins and denaturants. We use solubility data for a model tetraglycine peptide in each denaturant to tune the protein-denaturant interaction by a novel simulation methodology. We validate the results against data for more complex sequences: single-molecule Förster resonance energy transfer data for a 34-residue fragment of the globular protein CspTm and photoinduced electron transfer quenching data for the disordered peptides C(AGQ)nW in denaturant solution as well as the chemical denaturation of the mini-protein Trp cage. The combined force field model should aid our understanding of denaturation mechanisms and the interpretation of experiment.

Two Immunoglobulin Tandem Proteins with a Linking β-Strand Reveal Unexpected Differences in Cooperativity and Folding Pathways

The study of the folding of single domains, in the context of their multidomain environment, is important because more than 70% of eukaryotic proteins are composed of multiple domains. The structures of the tandem immunoglobulin (Ig) domain pairs A164–A165 and A168–A169, from the A-band of the giant muscle protein titin, reveal that they form tightly associated domain arrangements, connected by a continuous β-strand. We investigate the thermodynamic and kinetic properties of these tandem domain pairs. While A164–A165 apparently behaves as a single cooperative unit at equilibrium, unfolding without the accumulation of a large population of intermediates, domains in A168–A169 behave independently. Although A169 appears to be stabilized in the tandem protein, we show that this is due to nonspecific stabilization by extension. We elucidate the folding and unfolding pathways of both tandem pairs and show that cooperativity in A164–A165 is a manifestation of the relative refolding and unfolding rate constants of each individual domain. We infer that the differences between the two tandem pairs result from a different pattern of interactions at the domain/domain interface.