Heterogeneity in Protein Folding and Unfolding Reactions.

Proteins have dynamic structures that undergo chain motions on time scales spanning from picoseconds to seconds. Resolving the resultant conformational heterogeneity is essential for gaining accurate insight into fundamental mechanistic aspects of the protein folding reaction. The use of high-resolution structural probes, sensitive to population distributions, has begun to enable the resolution of site-specific conformational heterogeneity at different stages of the folding reaction. Different states populated during protein folding, including the unfolded state, collapsed intermediate states, and even the native state, are found to possess significant conformational heterogeneity. Heterogeneity in protein folding and unfolding reactions originates from the reduced cooperativity of various kinds of physicochemical interactions between various structural elements of a protein, and between a protein and solvent. Heterogeneity may arise because of functional or evolutionary constraints. Conformational substates within the unfolded state and the collapsed intermediates that exchange at rates slower than the subsequent folding steps give rise to heterogeneity on the protein folding pathways. Multiple folding pathways are likely to represent distinct sequences of structure formation. Insight into the nature of the energy barriers separating different conformational states populated during (un)folding can also be obtained by resolving heterogeneity.

Mapping Distinct Sequences of Structure Formation Differentiating Multiple Folding Pathways of a Small Protein.

To determine experimentally how the multiple folding pathways of a protein differ, in the order in which the structural parts are assembled, has been a long-standing challenge. To resolve whether structure formation during folding can progress in multiple ways, the complex folding landscape of monellin has been characterized, structurally and temporally, using the multisite time-resolved FRET methodology. After an initial heterogeneous polypeptide chain collapse, structure formation proceeds on parallel pathways. Kinetic analysis of the population evolution data across various protein segments provides a clear structural distinction between the parallel pathways. The analysis leads to a phenomenological model that describes how and when discrete segments acquire structure independently of each other in different subensembles of protein molecules. When averaged over all molecules, structure formation is seen to progress as α-helix formation, followed by core consolidation, then ß-sheet formation, and last end-to-end distance compaction. Parts of the protein that are closer in the primary sequence acquire structure before parts separated by longer sequence.

Site-specific time-resolved FRET reveals local variations in the unfolding mechanism in an apparently two-state protein unfolding transition.

Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the ß-sheet.

Graded Structural Polymorphism in a Bacterial Thermosensor Protein.

Thermosensing is critical for the expression of virulence genes in pathogenic bacteria that infect warm-blooded hosts. Proteins of the Hha-family, conserved among enterobacteriaceae, have been implicated in dynamically regulating the expression of a large number of genes upon temperature shifts. However, there is little mechanistic evidence at the molecular level as to how changes in temperature are transduced into structural changes and hence the functional outcome. In this study, we delineate the conformational behavior of Cnu, a putative molecular thermosensor, employing diverse spectroscopic, calorimetric and hydrodynamic measurements. We find that Cnu displays probe-dependent unfolding in equilibrium, graded increase in structural fluctuations and temperature-dependent swelling of the dimensions of its native ensemble within the physiological range of temperatures, features that are indicative of a highly malleable native ensemble. Site-specific fluorescence and NMR experiments in combination with multiple computational approaches-statistical mechanical model, coarse-grained and all-atom MD simulations-reveal that the fourth helix of Cnu acts as a unique thermosensing module displaying varying degrees of order and orientation in response to temperature modulations while undergoing a continuous unfolding transition. Our combined experimental-computational study unravels the folding-functional landscape of a natural thermosensor protein, the molecular origins of its unfolding complexity, highlights the role of functional constraints in determining folding-mechanistic behaviors, and the design principles orchestrating the signal transduction roles of the Hha protein family.

Understanding the heterogeneity intrinsic to protein folding.

Relating the native fold of a protein to its amino acid sequence remains a fundamental problem in biology. While computer algorithms have demonstrated recently their prowess in predicting what structure a particular amino acid sequence will fold to, an understanding of how and why a specific protein fold is achieved remains elusive. A major challenge is to define the role of conformational heterogeneity during protein folding. Recent experimental studies, utilizing time-resolved FRET, hydrogen-exchange coupled to mass spectrometry, and single-molecule force spectroscopy, often in conjunction with simulation, have begun to reveal how conformational heterogeneity evolves during folding, and whether an intermediate ensemble of defined free energy consists of different sub-populations of molecules that may differ significantly in conformation, energy and entropy.

Resolving Site-Specific Heterogeneity of the Unfolded State under Folding Conditions.

Understanding the properties of the unfolded state under folding conditions is of fundamental importance for gaining mechanistic insight into folding as well as misfolding reactions. Toward achieving this objective, the folding reaction of a small protein, monellin, has been resolved structurally and temporally, with the use of the multisite time-resolved FRET methodology. The present study establishes that the initial polypeptide chain collapse is not only heterogeneous but also structurally asymmetric and nonuniform. The population-averaged size for the segments spanning parts of the ß-sheet decreases much more than that for the α-helix. Multisite measurements enabled specific and nonspecific components of the initial chain collapse to be discerned. The expanded and compact intermediate subensembles have the properties of a nonspecifically collapsed (hence, random-coil-like) and specifically collapsed (hence, globular) polymer, respectively. During subsequent folding, both the subensembles underwent contraction to varying extents at the four monitored segments, which was close to gradual in nature. The expanded intermediate subensemble exhibited an additional very slow contraction, suggestive of the presence of non-native interactions that result in a higher effective viscosity slowing down intrachain motions under folding conditions.

α-Synuclein aggregation nucleates through liquid-liquid phase separation.

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson's disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid-liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson's disease mutations, also promote α-Syn liquid-liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson's disease pathogenesis.

Observation of Continuous Contraction and a Metastable Misfolded State during the Collapse and Folding of a Small Protein.

To obtain proper insight into how structure develops during a protein folding reaction, it is necessary to understand the nature and mechanism of the polypeptide chain collapse reaction, which marks the initiation of folding. Here, the time-resolved fluorescence resonance energy transfer technique, in which the decay of the fluorescence light intensity with time is used to determine the time evolution of the distribution of intra-molecular distances, has been utilized to study the folding of the small protein, monellin. It is seen that when folding begins, about one-third of the protein molecules collapse into a molten globule state (IMG), from which they relax by continuous further contraction to transit to the native state. The larger fraction gets trapped into a metastable misfolded state. Exit from this metastable state occurs via collapse to the lower free energy IMG state. This exit is slow, on a time scale of seconds, because of activation energy barriers. The trapped misfolded molecules as well as the IMG molecules contract continuously and slowly as structure develops. A phenomenological model of Markovian evolution of the polymer chain undergoing folding, incorporating these features, has been developed, which fits well the experimentally observed time evolution of distance distributions. The observation that the "wrong turn" to the misfolded state has not been eliminated by evolution belies the common belief that the folding of functional protein sequences is very different from that of a random heteropolymer, and the former have been selected by evolution to fold quickly.