A small single-domain protein folds through the same pathway on and off the ribosome.

In vivo, proteins fold and function in a complex environment subject to many stresses that can modulate a protein's energy landscape. One aspect of the environment pertinent to protein folding is the ribosome, since proteins have the opportunity to fold while still bound to the ribosome during translation. We use a combination of force and chemical denaturant (chemomechanical unfolding), as well as point mutations, to characterize the folding mechanism of the src SH3 domain both as a stalled ribosome nascent chain and free in solution. Our results indicate that src SH3 folds through the same pathway on and off the ribosome. Molecular simulations also indicate that the ribosome does not affect the folding pathway for this small protein. Taken together, we conclude that the ribosome does not alter the folding mechanism of this small protein. These results, if general, suggest the ribosome may exert a bigger influence on the folding of multidomain proteins or protein domains that can partially fold before the entire domain sequence is outside the ribosome exit tunnel.

Basis of Protein Stabilization by K Glutamate: Unfavorable Interactions with Carbon, Oxygen Groups.

Potassium glutamate (KGlu) is the primary Escherichia coli cytoplasmic salt. After sudden osmotic upshift, cytoplasmic KGlu concentration increases, initially because of water efflux and subsequently by K+ transport and Glu- synthesis, allowing water uptake and resumption of growth at high osmolality. In vitro, KGlu ranks with Hofmeister salts KF and K2SO4 in driving protein folding and assembly. Replacement of KCl by KGlu stabilizes protein-nucleic acid complexes. To interpret and predict KGlu effects on protein processes, preferential interactions of KGlu with 15 model compounds displaying six protein functional groups-sp3 (aliphatic) C; sp2 (aromatic, amide, carboxylate) C; amide and anionic (carboxylate) O; and amide and cationic N-were determined by osmometry or solubility assays. Analysis of these data yields interaction potentials (α-values) quantifying non-Coulombic chemical interactions of KGlu with unit area of these six groups. Interactions of KGlu with the 15 model compounds predicted from these six α-values agree well with experimental data. KGlu interactions with all carbon groups and with anionic (carboxylate) and amide oxygen are unfavorable, while KGlu interactions with cationic and amide nitrogen are favorable. These α-values, together with surface area information, provide quantitative predictions of why KGlu is an effective E. coli cytoplasmic osmolyte (because of the dominant effect of unfavorable interactions of KGlu with anionic and amide oxygens and hydrocarbon groups on the water-accessible surface of cytoplasmic biopolymers) and why KGlu is a strong stabilizer of folded proteins (because of the dominant effect of unfavorable interactions of KGlu with hydrocarbon groups and amide oxygens exposed in unfolding).

Probing the protein-folding mechanism using denaturant and temperature effects on rate constants.

Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high-free-energy transition state (TS) will help answer some of these. Here, we use effects of denaturants (urea, guanidinium chloride) and temperature on folding and unfolding rate constants and the overall equilibrium constant as probes of surface area changes in protein folding. We interpret denaturant kinetic m-values and activation heat capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding. Predicted accessible surface area changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50-90% of overall surface burial) and that the surface buried is disproportionately amide, demonstrating extensive formation of secondary structure in early intermediates. Models of possible pre-TS intermediates with all elements of the native secondary structure, created for several of these proteins, bury less amide and hydrocarbon surface than predicted for TS. Therefore, we propose that TS generally has both the native secondary structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.

A Computational Re-examination of the Criegee Intermediate-Sulfur Dioxide Reaction.

The atmospheric oxidation of sulfur dioxide by the parent and dimethyl Criegee intermediates (CIs) may be an important source of sulfuric acid aerosol, which has a large impact on radiative forcing and therefore upon climate. A number of computational studies have considered how the CH2OOS(O)O heteroozonide (HOZ) adduct formed in the CI + SO2 reaction converts SO2 to SO3. In this work we use the CBS-QB3 quantum chemical method along with equation-of-motion spin-flip CCSD(dT) and MCG3 theories to reveal new details regarding the formation and decomposition of the endo and exo conformers of the HOZ. Although ∼75% of the parent CI + SO2 reaction is initiated by formation of the exo HOZ, hyperconjugation preferentially stabilizes many of the endo intermediates and transition structures by 1-5 kcal mol(-1). Our quantum chemical calculations, in conjunction with statistical rate theory models, predict a rate coefficient for the parent CI + SO2 reaction of 3.68 × 10(-11) cm(3) molecule(-1) s(-1), in good agreement with recent experimental measurements. RRKM/master equation simulations based on our quantum chemical data predict a prompt carbonyl + SO3 yield of >95% for the reaction of both the parent and dimethyl CI with SO2. The existence of concerted cycloreversion transition structures 10-15 kcal mol(-1) higher in energy than the HOZ accounts for most of the predicted SO3 formation.

Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer.

To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients K(p) for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. K(p) values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH--amide O and amide NH--amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or K(p) values.

Quantifying additive interactions of the osmolyte proline with individual functional groups of proteins: comparisons with urea and glycine betaine, interpretation of m-values.

To quantify interactions of the osmolyte l-proline with protein functional groups and predict their effects on protein processes, we use vapor pressure osmometry to determine chemical potential derivatives dµ2/dm3 = µ23, quantifying the preferential interactions of proline (component 3) with 21 solutes (component 2) selected to display different combinations of aliphatic or aromatic C, amide, carboxylate, phosphate or hydroxyl O, and amide or cationic N surface. Solubility data yield µ23 values for four less-soluble solutes. Values of µ23 are dissected using an ASA-based analysis to test the hypothesis of additivity and obtain α-values (proline interaction potentials) for these eight surface types and three inorganic ions. Values of µ23 predicted from these α-values agree with the experiment, demonstrating additivity. Molecular interpretation of α-values using the solute partitioning model yields partition coefficients (Kp) quantifying the local accumulation or exclusion of proline in the hydration water of each functional group. Interactions of proline with native protein surfaces and effects of proline on protein unfolding are predicted from α-values and ASA information and compared with experimental data, with results for glycine betaine and urea, and with predictions from transfer free energy analysis. We conclude that proline stabilizes proteins because of its unfavorable interactions with (exclusion from) amide oxygens and aliphatic hydrocarbon surfaces exposed in unfolding and that proline is an effective in vivo osmolyte because of the osmolality increase resulting from its unfavorable interactions with anionic (carboxylate and phosphate) and amide oxygens and aliphatic hydrocarbon groups on the surface of cytoplasmic proteins and nucleic acids.

Quantifying functional group interactions that determine urea effects on nucleic acid helix formation.

Urea destabilizes helical and folded conformations of nucleic acids and proteins, as well as protein-nucleic acid complexes. To understand these effects, extend previous characterizations of interactions of urea with protein functional groups, and thereby develop urea as a probe of conformational changes in protein and nucleic acid processes, we obtain chemical potential derivatives (µ23 = dµ2/dm3) quantifying interactions of urea (component 3) with nucleic acid bases, base analogues, nucleosides, and nucleotide monophosphates (component 2) using osmometry and hexanol-water distribution assays. Dissection of these µ23 values yields interaction potentials quantifying interactions of urea with unit surface areas of nucleic acid functional groups (heterocyclic aromatic ring, ring methyl, carbonyl and phosphate O, amino N, sugar (C and O); urea interacts favorably with all these groups, relative to interactions with water. Interactions of urea with heterocyclic aromatic rings and attached methyl groups (as on thymine) are particularly favorable, as previously observed for urea-homocyclic aromatic ring interactions. Urea m-values determined for double helix formation by DNA dodecamers near 25 °C are in the range of 0.72-0.85 kcal mol(-1)m(-1) and exhibit little systematic dependence on nucleobase composition (17-42% GC). Interpretation of these results using the urea interaction potentials indicates that extensive (60-90%) stacking of nucleobases in the separated strands in the transition region is required to explain the m-value. Results for RNA and DNA dodecamers obtained at higher temperatures, and literature data, are consistent with this conclusion. This demonstrates the utility of urea as a quantitative probe of changes in surface area (ΔASA) in nucleic acid processes.

Role of nearshore benthic algae in the Lake Michigan silica cycle.

Si cycling is linked with processes from global carbon sequestration to community composition and is especially important in aquatic ecosystems. Lake Michigan has seen dramatic fluctuations in dissolved silica (dSi) over several decades, which have been examined in the context of planktonic processes (diatom blooms), but the role of benthic organisms (macroalgae and their epiphytes) in Si cycling have not been explored. To assess significance of nearshore benthic algae in Si dynamics, we assembled dSi data from an offshore site sampled since the late 1980's, and sampled off three Milwaukee beaches during 2005-19. Using colorimetric assays and alkaline digestion, we measured dSi, biogenic silica in particulate suspended material (pSi) and biogenic silica in benthic macroalgae (Cladophora) and epiphytic diatoms (bSi). Offshore, dSi increased about 1 µM per year from 25 µM in the late 1980's to nearly 40 µM in 2019. Nearshore dSi fluctuated dramatically annually, from near zero to concentrations similar to offshore. Both Cladophora and its epiphytes contained significant bSi, reaching up to 30% of dry mass (300 mg Si g dry mass-1) of the assemblage in summer. Microscopic analyses including localization with a Si-specific-stain and X-ray microanalysis showed bSi in epiphytic diatom cells walls, but the nature and localization of Si in macroalgae remained unclear. A simple model was developed estimating Si demand of algae using the areal macroalgal biomass, growth rates inferred from P-content, and bSi content, and comparing Si demand with dSi available in the water column. This indicated that 7-70% of the dSi in water overlying nearshore benthic algal beds could be removed per day. Key elements of the Si cycle, including which organisms sequester bSi and how rapidly Si is recycled, remain unclear. This work has implications for coastal marine waters where large macroalgal biomass accumulates but bSi content is virtually unknown.

Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding.

While single-molecule force spectroscopy has greatly advanced the study of protein folding, there are limitations to what can be learned from studying the effect of force alone. We developed a novel technique, chemo-mechanical unfolding, that combines multiple perturbants-force and chemical denaturant-to more fully characterize the folding process by simultaneously probing multiple structural parameters-the change in end-to-end distance, and solvent accessible surface area. Here, we describe the theoretical background, experimental design, and data analysis for chemo-mechanical unfolding experiments probing protein folding thermodynamics and kinetics. This technique has been applied to characterize parallel protein folding pathways, the protein denatured state, protein folding on the ribosome, and protein folding intermediates.

Exploring the Denatured State Ensemble by Single-Molecule Chemo-Mechanical Unfolding: The Effect of Force, Temperature, and Urea.

While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the Tm and Cm. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.

Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein.

A fundamental question in protein folding is whether proteins fold through one or multiple trajectories. While most experiments indicate a single pathway, simulations suggest proteins can fold through many parallel pathways. Here, we use a combination of chemical denaturant, mechanical force and site-directed mutations to demonstrate the presence of multiple unfolding pathways in a simple, two-state folding protein. We show that these multiple pathways have structurally different transition states, and that seemingly small changes in protein sequence and environment can strongly modulate the flux between the pathways. These results suggest that in vivo, the crowded cellular environment could strongly influence the mechanisms of protein folding and unfolding. Our study resolves the apparent dichotomy between experimental and theoretical studies, and highlights the advantage of using a multipronged approach to reveal the complexities of a protein's free-energy landscape.