Combining Ultrarapid Mixing with Photochemical Oxidation to Probe Protein Folding

We demonstrate a new method to study protein folding by combining fast photochemical oxidation of proteins (FPOP) with ultrarapid microfluidic mixing to observe kinetics on the microsecond time scale. Folding proteins pass through a focused UV laser beam, creating OH radicals that label the select protein side chains and are analyzed with mass spectrometry. As a proof of principle, we demonstrate this method with hen egg lysozyme that shows at least two kinetic phases before 1 ms, which are compared with those observed by Trp fluorescence. This method provides another, complementary probe of the early, complex steps of protein folding

Modeling Concentration-dependent Phase Separation Processes Involving Peptides and RNA via Residue-Based Coarse-Graining

Biomolecular condensation, especially liquid–liquid phase separation, is an important physical process with relevance for a number of different aspects of biological functions. Key questions of what drives such condensation, especially in terms of molecular composition, can be addressed via computer simulations, but the development of computationally efficient yet physically realistic models has been challenging. Here, the coarse-grained model COCOMO is introduced that balances the polymer behavior of peptides and RNA chains with their propensity to phase separate as a function of composition and concentration. COCOMO is a residue-based model that combines bonded terms with short- and long-range terms, including a Debye–Hückel solvation term. The model is highly predictive of experimental data on phase-separating model systems. It is also computationally efficient and can reach the spatial and temporal scales on which biomolecular condensation is observed with moderate computational resources

Effects of Mutations on the Reconfiguration Rate of α‑Synuclein

It is still poorly understood why α-synuclein, the intrinsically disordered protein involved in Parkinson’s and other neurodegenerative diseases, is so prone to aggregation. Recent work has shown a correlation between the aggregation rate and the rate of diffusional reconfiguration by varying temperature and pH. Here we examine the effects of several point mutations in the sequence on the conformational ensemble and reconfiguration rate. We find that at lower temperatures the PD causing aggregation enhancing mutations slow down and aggregation reducing mutations drastically speed up intramolecular diffusion, as compared to the wild type sequence. However, at higher temperatures, one of three familial mutations that enhance aggregation slows intramolecular diffusion while non-natural mutations that inhibit aggregation speed up intramolecular diffusion. These results support the hypothesis that the first step of aggregation is kinetically controlled by reconfiguration in which the protein chain cannot reconfigure rapidly enough to escape oligomerization. Finally we provide physical and chemical insights into why small point mutations cause these dramatic changes in the conformational ensemble and dynamics

Sub-millisecond Chain Collapse of the Escherichia coli Globin ApoHmpH

Myoglobins are ubiquitous proteins that play a seminal role in oxygen storage, transport, and NO metabolism. The folding mechanism of apomyoglobins from different species has been studied to a fair extent over the last two decades. However, integrated investigations of the entire process, including both the early (sub-ms) and late (ms–s) folding stages, have been missing. Here, we study the folding kinetics of the single-Trp <i>Escherichia coli</i> globin apoHmpH via a combination of continuous-flow microfluidic and stopped-flow approaches. A rich series of molecular events emerges, spanning a very wide temporal range covering more than 7 orders of magnitude, from sub-microseconds to tens of seconds. Variations in fluorescence intensity and spectral shifts reveal that the protein region around Trp₁₂₀ undergoes a fast collapse within the 8 μs mixing time and gradually reaches a native-like conformation with a half-life of 144 μs from refolding initiation. There are no further fluorescence changes beyond ca. 800 μs, and folding proceeds much more slowly, up to 20 s, with acquisition of the missing helicity (ca. 30%), long after consolidation of core compaction. The picture that emerges is a gradual acquisition of native structure on a free-energy landscape with few large barriers. Interestingly, the single tryptophan, which lies within the main folding core of globins, senses some local structural consolidation events after establishment of native-like core polarity (i.e., likely after core dedydration). In all, this work highlights how the main core of the globin fold is capable of becoming fully native efficiently, on the sub-millisecond time scale

Exploring the Energy Landscape of Nucleic Acid Hairpins Using Laser Temperature-Jump and Microfluidic Mixing

We have investigated the multidimensionality of the free energy landscape accessible to a nucleic acid hairpin by measuring the relaxation kinetics in response to two very different perturbations of the folding/unfolding equilibrium, either a laser temperature-jump or ion-jump (from rapid mixing with counterions). The two sets of measurements carried out on DNA hairpins (4 or 5 base pairs in the stem and 21-nucleotide polythymine loop), using FRET between end labels or fluorescence of 2-aminopurine in the stem as conformational probes, yield distinctly different relaxation kinetics in the temperature range 10–30 °C and salt range 100–500 mM NaCl, with rapid mixing exhibiting slower relaxation kinetics after an initial collapse of the chain within 8 μs of the counterion mixing time. The discrepancy in the relaxation times increases with increasing temperatures, with rapid mixing times nearly 10-fold slower than T-jump times at 30 °C. These results rule out a simple two-state scenario with the folded and unfolded ensemble separated by a significant free energy barrier, even at temperatures close to the thermal melting temperature <i>T</i>_m. Instead, our results point to the scenario in which the conformational ensemble accessed after counterion condensation and collapse of the chain is distinctly different from the unfolded ensemble accessed with T-jump perturbation. Our data suggest that, even at temperatures in the vicinity of <i>T</i>_m or higher, the relaxation kinetics obtained from the ion-jump measurements are dominated by the escape from the collapsed state accessed after counterion condensation