Dry Molten Research Articles

Unfolding of a Small Protein Proceeds via Dry and Wet Globules and a Solvated Transition State

Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N∗. Fluorescence resonance energy transfer measurements show that N∗ then expands rapidly but partially to form an early unfolding intermediate IE. Fluorescence spectral measurements indicate that both N∗ and IE have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, IL, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when IL forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.

Packing in molten globules and native states

Close packing of hydrophobic residues in the protein interior is an important determinant of protein stability. Cavities introduced by large to small substitutions are known to destabilize proteins. Conversely, native states of proteins and protein fragments can be stabilized by filling in existing cavities. Molten globules (MGs) were initially used to describe a state of protein which has well-defined secondary structure but little or no tertiary packing. Subsequent studies have shown that MGs do have some degree of native-like topology and specific packing. Wet molten globules (WMGs) with hydrated cores and considerably decreased packing relative to the native state have been studied extensively. Recently there has been renewed interest in identification and characterization of dry molten globules (DMGs). These are slightly expanded forms of the native state which show increased conformational flexibility, native-like main-chain hydrogen bonding and dry interiors. The generality of occurrence of DMGs during protein unfolding and the extent and nature of packing in DMGs remain to be elucidated. Packing interactions in native proteins and MGs can be probed through mutations. Next generation sequencing technologies make it possible to determine relative populations of mutants in a large pool. When this is coupled to phenotypic screens or cell-surface display, it becomes possible to rapidly examine large panels of single-site or multi-site mutants. From such studies, residue specific contributions to protein stability and function can be estimated in a highly parallelized fashion. This complements conventional biophysical methods for characterization of packing in native states and molten globules.

Molten globules, entropy-driven conformational change and protein folding

The exquisite side chain close-packing in the protein core and at binding interfaces has prompted a conviction that packing selectivity is the primary mechanism for molecular recognition in folding and/or binding reactions. Contrary to this view, molten globule proteins can adopt native topology and bind targets tightly and specifically in the absence of side chain close-packing. The molten globule is a highly dynamic form with native-like secondary structure and a loose protein core that admits solvent. The related (but still controversial) dry molten globule is an expanded form of the native protein with largely intact topology but a tighter protein core that excludes solvent. Neither form retains side chain close-packing, and therefore both structure and function must result from other factors, assuming that the reality of the dry molten globule is accepted. This simplifying realization calls for a re-evaluation of established models.

Coupled motion in proteins revealed by pressure perturbation.

The cooperative nature of protein substructure and internal motion is a critical aspect of their functional competence about which little is known experimentally. NMR relaxation is used here to monitor the effects of high pressure on fast internal motion in the protein ubiquitin. In contrast to the main chain, the motions of the methyl-bearing side chains have a large and variable pressure dependence. Within the core, this pressure sensitivity correlates with the magnitude of motion at ambient pressure. Spatial clustering of the dynamic response to applied hydrostatic pressure is also seen, indicating localized cooperativity of motion on the sub-nanosecond time scale and suggesting regions of variable compressibility. These and other features indicate that the native ensemble contains a significant fraction of members with characteristics ascribed to the recently postulated "dry molten globule". The accompanying variable side-chain conformational entropy helps complete our view of the thermodynamic architecture underlying protein stability, folding, and function.

Dry molten globule intermediates and the mechanism of protein unfolding

New experimental results show that either gain or loss of close packing can be observed as a discrete step in protein folding or unfolding reactions. This finding poses a significant challenge to the conventional two-state model of protein folding. Results of interest involve dry molten globule (DMG) intermediates, an expanded form of the protein that lacks appreciable solvent. When an unfolding protein expands to the DMG state, side chains unlock and gain conformational entropy, while liquid-like van der Waals interactions persist. Four unrelated proteins are now known to form DMGs as the first step of unfolding, suggesting that such an intermediate may well be commonplace in both folding and unfolding. Data from the literature show that peptide amide protons are protected in the DMG, indicating that backbone structure is intact despite loss of side-chain close packing. Other complementary evidence shows that secondary structure formation provides a major source of compaction during folding. In our model, the major free-energy barrier separating unfolded from native states usually occurs during the transition between the unfolded state and the DMG. The absence of close packing at this barrier provides an explanation for why phi-values, derived from a Brønsted-Leffler plot, depend primarily on structure at the mutational site and not on specific side-chain interactions. The conventional two-state folding model breaks down when there are DMG intermediates, a realization that has major implications for future experimental work on the mechanism of protein folding.

An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain

A reversible structural unlocking reaction, in which the close-packed van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (DeltaH(0) = 35 +/- 4 kJ/mol) which is nearly compensated in free energy by the entropy change (DeltaS(0) = 112 +/- 20 Jxmol(-1)xK(-1)). The unlocking reaction has a time constant of about 1 mus at 5 degrees C and is enthalpy-limited with an activation energy of 32 +/- 1 kJ/mol and a large Arrhenius preexponential factor of A = 7.5 x 10(11) s(-1). In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 +/- 1 kJ/mol leads to unfolding of the C-terminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the C-terminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.

Direct evidence for a dry molten globule intermediate during the unfolding of a small protein

Little is known about how proteins begin to unfold. In particular, how and when water molecules penetrate into the protein interior during unfolding, thereby enabling the dissolution of specific structure, is poorly understood. The hypothesis that the native state expands initially into a dry molten globule, in which tight packing interactions are broken, but whose hydrophobic core has not expanded sufficiently to be able to absorb water molecules, has very little experimental support. Here, we report our analysis of the earliest observable events during the unfolding of single chain monellin (MNEI), a small plant protein. Far- and near-UV circular dichroism measurements of GdnHCl-induced unfolding indicate that a molten globule intermediate forms initially, before the major slow unfolding reaction commences. Steady-state fluorescence resonance energy transfer measurements show that the C-terminal end of the single helix of MNEI initially moves rapidly away from the single tryptophan residue that is close to the N-terminal end of the helix. The average end-to-end distance of the protein also expands during unfolding to the molten globule intermediate. At this time, water has yet to penetrate the protein core, according to the evidence from intrinsic tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid fluorescence-monitored kinetic unfolding measurements. Our results therefore provide direct evidence for a dry molten globule intermediate at the initial stage of unfolding. Our results further suggest that the structural transition between the native and dry molten globule states could be an all-or-none transition, whereas further swelling of the globule appears to occur gradually.

Ceramic anode catalyst for dry methane type molten carbonate fuel cell

Abstract Oxide catalyst materials for methane oxidation were examined in order to develop the anode electrode for molten carbonate type fuel cell (MCFC). As a primary selection, oxides such as lanthanum (La 2 O 3 ) and samarium (Sm 2 O 3 ) were selected from screening experiments of TPD, TG and tubular reactor. Composite materials of these oxides with titanium fine powder were assembled into a cell unit for MCFC as the anode electrode. Steady-state activities were observed with these anode electrode materials when hydrogen was used as a fuel. When methane was directly charged to anode as a fuel (dry methane operation), a power generation with steady state was observed on both lanthanum and samarium composites after gradual decrease of open circuit electromotive force (OCV) and closed circuit current (CCI). The steady-state activity held as long as 144 h of continuous operation.

Mechanism of formation of a productive molten globule form of barstar.

Structural analysis of the initial steps in protein folding is difficult because of the swiftness with which these steps occur. Hence, the link between initial polypeptide chain collapse and formation of secondary and other specific structures remains poorly understood. Here, an equilibrium model has been developed for characterizing the initial steps of folding of the small protein barstar, which lead to the formation of a productive molten globule in the folding pathway. In this model, the high-pH-unfolded form (D form) of barstar, which is shown to be as unstructured as the urea-denatured form, is transformed progressively into a molten globule B form by incremental addition of the salt Na(2)SO(4) at pH 12. At very low concentrations of Na(2)SO(4), the D form collapses into a pre-molten globule (P) form, whose volume exceeds that of the native (N) state by only 20%, and which lacks any specific structure as determined by far- and near-UV circular dichroism. At higher concentrations of Na(2)SO(4), the P form transforms into the molten globule (B) form in a highly noncooperative transition populated by an ensemble of at least two intermediates. The B form is a dry molten globule in which water is excluded from the core, and in which secondary structure develops to 65% and tertiary contacts develop to 40%, relative to that of the native protein. Kinetic refolding experiments carried out at pH 7 and at high Na(2)SO(4) concentrations, in which the rate of folding of the D form to the N state is compared to that of the B form to the N state, indicate conclusively that the B form is a productive intermediate that forms on the direct pathway of folding from the D form to the N state.

Synthesis of radioiodinated 4-(*I)iodoantipyrine via isotopic exchange

Radioiodinated 4-[*I]iodoantipyrine labeled with radioiodine (i.e., 123I or 125I or 131I) has been used for modeling radiation damage on cell nuclei of tumor cells where the characteristic high linear energy transfer (high-LET) of the Auger electron could be demonstrated. Also, the compound is currently used for the measurement of regional cerebral blood flow (rCBF) in autoradiography. 4-[131I]iodoantipyrine was synthesized by two methods via a nucleophilic isotopic exchange reaction between 131I as iodide ion [131I–] and inactive 4-[127I]iodoantipyrine: either in absolute ethyl alcohol catalyzed by ammonium acetate or in dry state molten ammonium acetate (m.p. 114 °C) as an isotopic exchange medium without carrier addition. The first one is called wet method: where a solution of 4-iodoantipyrine and ammonium acetate in absolute ethyl alcohol and lyophilized Na131I was heated briefly up to boiling (80 to 90 °C) for 30 minutes under reflux. The second one is called dry state-molten method: where the alcoholic solution containing 4-iodoantipyrine and ammonium acetate and the lyophilized Na131I were heated briefly in a nitrogen stream to dryness at 120 to 125 °C for 5 minutes or melted by gradual heating at 150 to 160 °C for 5 minutes. A radiochemical yield ranged between 90%–95% in each method has been obtained for 4-[131I]iodoantipyrine. In both methods, the reaction proceeds properly without carrier addition by an addition – elimination mechanism. The physico-chemical parameters affecting the radiochemical yield of the isotopic exchange reaction [i.e., reaction time, temperature, exchange medium, concentration of the reactants, carrier (KI) addition and pH] were investigated. Chromatographic analysis i.e., TLC and HPLC were used to determine the radiochemical yield as well as the purity of the final product, which was as pure as 99.9%.

The kinetic basis for the stabilization of staphylococcal nuclease by xylose.

The effect of xylose on the rates of folding and unfolding of staphylococcal nuclease (nuclease) have been investigated using fluorescence-detected pressure-jump relaxation kinetics in order to establish the kinetic basis for the observed stabilization of nuclease by this sugar (Frye KJ, Perman CS, Royer CA, 1996, Biochemistry 35:10234-10239). The activation volumes for both folding and unfolding and the equilibrium volume change for folding were all positive. Their values were within experimental error of those reported previously (Vidugiris GJA, Markley JL, Royer CA, 1995, Biochemistry 34:4909-4912) and were independent of xylose concentration. The major effect of xylose concentration was to increase significantly the rate of folding. The large positive activation volume for folding was interpreted previously as indicating that the rate-limiting step in nuclease folding involves dehydration of a significant amount of surface area. A large effect of xylose on the rate constant for folding provides strong support for this interpretation, because xylose, an osmolyte, stabilizes the folded state of proteins through surface tension effects. These studies further characterize the transition state in nuclease folding as lying closer to the folded, rather than the unfolded state along the folding coordinate in terms of the degree of burial of surface area. The image of the transition state that emerges is consistent with a dry molten globule.

Direct NMR evidence for an intermediate preceding the rate-limiting step in the unfolding of ribonuclease A.

It is commonly believed that there are no detectable intermediates in the kinetic unfolding reactions of small proteins. If such intermediates could be found, they would give important information about the nature of the transition state for unfolding, which is thought to occur close to the native state. We report here that one-dimensional proton magnetic resonance spectra recorded during the unfolding of ribonuclease A provide direct evidence for at least one unfolding intermediate in which side chains are free to rotate. This intermediate appears to be a 'dry molten globule' of the kind hypothesized by Shakhnovich and Finkelstein.

Diffusion of dissolved CO 2 and Cl in hydrous silicic to intermediate magmas

The bulk diffusivity of dissolved CO 2 in obsidian and dacite melts containing 0–11wt% dissolved water at 800–1100°C and 1 GPa was characterized using a 14C radiotracer technique. Five values were also obtained for Cl in melts containing 8% H 2O. Carbon dioxide diffusivity ( D CO 2 ) in nominally dry molten obsidian is similar to previously reported values for a simple melt in the Na 2O-Al 2O 3-SiO 2 system and for dry basalt, indicating little effect of melt composition on bulk CO 2 diffusion. Three D CO 2 values for hydrous dacite melt are consistent with this lack of a melt composition effect. Dissolved water, however, causes an increase of D CO 2 in molten obsidian by about an order of magnitude for each 5% of added water, such that at 8 wt% H 2O D CO 2 = (6.5 × 10 −4) exp( −18000 ± 5000 RT ) where D CO 2 is in cm 2/s and R in cal/deg-mol. The activation energy of ~ 18 kcal/mol is lower by a factor of ~3 than that for diffusion in dry basalt. In comparison with existing data for diffusion of water in silicic melts at their typical liquidus temperatures, D CO 2 is generally lower unless the melt contains more than about 6% dissolved H 2O, in which case D CO 2 ~ D H 2O . The few data obtained for chlorine diffusion reveal that D Cl is lower by a factor of 5–10 than D CO 2 in the same melt. The overall systematics of the new data on dissolved volatile diffusion indicate that diffusional fractionation effects are likely to be most significant in silicic to intermediate magmas having water contents in the 0–4% range.

Oxygen depolarized electrolysis of sodium chloride by the use of a ?-alumina-molten salt system

This paper is concerned with oxygen depolarized electrolysis of NaCl by the use ofβ-alumina solid electrolyte and molten salts. In the electrolysis, dry chlorine gas and pure molten sodium hydroxide are produced from sodium chloride, oxygen and water. Because the oxygen reduction proceeds very smoothly in the molten sodium hydroxide, this process is very promising for the future. The theoretical decomposition voltage of this process is estimated to be 1.5 V, which is lower than that of the process without an oxygen cathode by 1.1 V. The model cell study shows that a terminal voltage of 3 V at 50 A dm−2 is attainable.

Mechanism of Chloride Stress Corrosion Cracking of Austenitic Stainless Steels

Abstract Electrochemical studies were made in aqueous LiCl, MgCl2, and MgBr2 solutions and in ZnCl2/KCl molten salt to clarify the corrosion reactions related to stress corrosion cracking (SCC) of austenitic stainless steel and to better define environmental variables critical to the occurrence of chloride SCC. Type 304 stainless steel electrodes were employed, with complementary SCC tests made with U-bend Type 304 stainless steel specimens. Several conclusions critical to an understanding of the mechanism of chloride SCC resulted from these investigations: (1) SCC was observed in concentrated MgBr2 solutions, (2) H2O must be present in the electrolyte, as SCC did not occur in dry molten ZnCl2/KCl, and (3) H2 evolution from corroding specimens may be facilitated by anodic polarization. Present studies do not support a model equating crack propagation with stress assisted anodic dissolution. Rather, evidence is presented that hydrogen evolution at the crack tip occurs and is a critical precursor to crack initiation and propagation. A model of SCC requiring hydrogen evolution at the crack tip is proposed, with emphasis being placed on the effect of anodic reactions within the crack in maintaining high acidity near the crack tip. Recent publications suggest that the role of evolved hydrogen in SCC may be related to formation of hydrogen induced martensitic platelets along paths of crack propagation.