Disulfide bond formation involves a quinhydrone-type charge-transfer complex

The chemistry of disulfide exchange in biological systems is well studied. However, the detailed mechanism of how oxidizing equivalents are derived to form disulfide bonds in proteins is not clear. In prokaryotic organisms, it is known that DsbB delivers oxidizing equivalents through DsbA to secreted proteins. DsbB becomes reoxidized by reducing quinones that are part of the membrane-bound electron-transfer chains. It is this quinone reductase activity that links disulfide bond formation to the electron transport system. We show here that purified DsbB contains the spectral signal of a quinhydrone, a charge-transfer complex consisting of a hydroquinone and a quinone in a stacked configuration. We conclude that disulfide bond formation involves a stacked hydroquinone-benzoquinone pair that can be trapped on DsbB as a quinhydrone charge-transfer complex. Quinhydrones are known to be redox-active and are commonly used as redox standards, but, to our knowledge, have never before been directly observed in biological systems. We also show kinetically that this quinhydrone-type charge-transfer complex undergoes redox reactions consistent with its being an intermediate in the reaction mechanism of DsbB. We propose a simple model for the action of DsbB where a quinhydrone-like complex plays a crucial role as a reaction intermediate.     

Disulfide bond formation by exported glutaredoxin indicates glutathione's presence in the E. coli periplasm

Organisms have evolved elaborate systems that ensure the homeostasis of the thiol redox environment in their intracellular compartments. In Escherichia coli, the cytoplasm is kept under reducing conditions by the thioredoxins with the help of thioredoxin reductase and the glutaredoxins with the small molecule glutathione and glutathione reductase. As a result, disulfide bonds are constantly resolved in this compartment. In contrast to the cytoplasm, the periplasm of E. coli is maintained in an oxidized state by DsbA, which is recycled by DsbB. Thioredoxin 1, when exported to the periplasm turns from a disulfide bond reductase to an oxidase that, like DsbA, is dependent on DsbB. In this study we set out to investigate whether a subclass of the thioredoxin superfamily, the glutaredoxins, can become disulfide bond-formation catalysts when they are exported to the periplasm. We find that glutaredoxins can promote disulfide bond formation in the periplasm. However, contrary to the behavior of thioredoxin 1 in this environment, the glutaredoxins do so independently of DsbB. Furthermore, we show that glutaredoxin 3 requires the glutathione biosynthesis pathway for its function and can oxidize substrates with only a single active-site cysteine. Our data provides in vivo evidence suggesting that oxidized glutathione is present in the E. coli periplasm in biologically significant concentrations.     

Disulfide bond dihedral angles from Raman spectroscopy

Raman spectra of several compounds containing the CS-SC moiety were obtained (in the solid phase) from 450-800 cm^-1 to investigate the S-S and C-S stretching behavior. The S-S stretching frequency varied linearly with the CS-SC dihedral angle (obtained from either x-ray or neutron diffraction or ultraviolet absorption) for compounds whose CC-SS dihedral angles were not very different. The ratio of the intensities of the S-S and C-S stretching bands exhibited no recognizable correlation with either the CS-SC dihedral angle or the CSS bond angle, probably because this ratio is sensitive to the crystalline environment. The linear dependence of the S-S stretching frequency on dihedral angle leads to a dihedral angle for the plant hormone, malformin A, that is in excellent agreement with that estimated from the longest wavelength CS-SC ultraviolet absorption band.     

Aging in the eye lens: Roles for proteolysis and nutrition in formation of cataract

Age-related eye lens opacification is one of the leading causes of blindess. Cataract afflicts a majority of persons over age 75, and costs associated with cataract treatment or extraction are the largest line-item in the Medicare budget. In the lesser-developed nations, the problem is more acute since cataract frequently occurs at younger ages, and there are insufficient numbers of ophthalmologists to perform lens extractions. In this paper, we examine causes of cataract and approaches to delay this debility. Lens proteins are damaged during aging, presumably by the light and oxygen to which they are exposed. In many cell types, damaged proteins are selectively and rapidly removed in part by cytoplasmic ATP-and ubiquitin-dependent systems. We hypothesized that the protein-editing capability exists in young lens tissue. However, upon aging, reduced proteolytic capability may be related to the accumulation of damaged proteins in cataracts. It is observed that lens tissue displays ATP-dependent proteolytic activity and ubiquitin-conjugating capability. Of the energy-dependent proteolytic capacity, 75% is dependent upon ubiquitin conjugation. Simulation of aging by ⁶⁰Co-irradiation of the major lens proteins, α-crystallin (0,0.1, 0.6, 2.6 mol hydroxyl radical per mole α-crystallin) caused both aggregation and lysis. Both are observed in the aged lens. Rates of degradation of differentially modified crystallin by lens proteasome indicated enhanced levels of proteolysis, with increasing photooxidation of substrate. Antioxidant nutrients, such as ascorbate, are found in eye tissues in relation to levels in diet in guinea pigs and humans. Elevated dietary ascorbate confers protection against solar-light-induced damage to lens proteins and proteases and may delay cataracts in animals subject to various stresses. Recent epidemiological evidence also indicates that persons with higher ascorbate status have diminished risk of various forms of cataract. Restricting dietary calorie intake in rodents delays cataract progress, extends life, prolongs immune function, diminishes incidence of cancer, etc. However, these benefits are not associated with elevated plasma levels of ascorbate. Symposium paper: Aging and Nutrition. Presented on October 5, 1990 during the 20th Annual Meeting of AGE in New York City.     

Antibody catalysis of peptide bond formation.

An antibody generated against a neutral phosphonate diester transition-state (TS not equal to) analog catalyzes the formation of an amide bond between a phenylalanyl amino group and an acyl azide derived from L-alanine. The antibody is selective for L- vs. D-alanine and does not catalyze the hydrolysis of the acyl azide to an appreciable degree. A rate acceleration of 10,000-fold relative to the uncatalyzed reaction is observed. The antibody may achieve its catalytic efficiency both by acting as an entropy trap and by stabilizing the deprotonated form of the amine nucleophile. These experiments constitute a first step toward a general strategy for the generation of sequence-specific peptide ligases.     

Sodium-23 magnetic resonance imaging of the eye and lens.

In order to develop a better understanding of cataract and to evaluate the effectiveness of potential drugs, noninvasive techniques must be devised to detect early metabolic changes. As a prelude to these goals, sodium-23 imaging experiments operating at 29.8 MHz (2.7 teslas) were performed on the bovine eye and lens. A spatially localized transverse relaxation time (T2)-weighted spin-density map of the sodium-23 within the lens is presented, with a resolution better than 250 micron. Due to the presence of short-T2 (3 msec) components within the lens, only the use of the planar-integral projection reconstruction (PPR) imaging scheme allowed sufficiently short echo-times (1 msec) to permit sodium-23 signal detection. These noninvasive imaging results show differences in the apparent sodium concentration within the lens that are consistent with separate, invasive measurements of sodium concentration. Separate analysis (with no spatial localization) at 79.4 MHz (7.2 teslas), using a shift reagent (dysprosium) to distinguish extracellular from intracellular sodium, indicates that approximately 62% of the detected sodium-23 signal is intracellular. These results are consistent with observations based on invasive measurements and further support the existence of the pump-leak system and a sodium gradient within the lens.     

A pathway for disulfide bond formation in vivo.

Protein disulfide bond formation in Escherichia coli requires the periplasmic protein DsbA. We describe here mutations in the gene for a second protein, DsbB, which is also necessary for disulfide bond formation. Evidence suggests that DsbB may act by reoxidizing DsbA, thereby regenerating its ability to donate its disulfide bond to target proteins. We propose that DsbB, an integral membrane protein, may be involved in transducing redox potential across the cytoplasmic membrane.     

A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens.

A discrete 10-kDa polypeptide (10K) is expressed from early stages in the embryonic chicken lens. Since this has potential as a marker for lens cell development, chicken 10K and its homologues from mouse and human lenses were identified by protein sequencing and cloning. Surprisingly, lens 10K proteins appear to be identical to a lymphokine, macrophage migration inhibitory factor (MIF), originally identified in activated human T cells. Using microdissection and PCR techniques, we find that expression of 10K/MIF is strongly correlated with cell differentiation in the developing chicken lens. Northern blot analysis shows that 10K/MIF is widely expressed in mouse tissues. These results suggest that proteins with MIF activity may have roles beyond the immune system, perhaps as intercellular messengers or part of the machinery of differentiation itself. Indeed, partial sequence of other small lens proteins identifies another MIF-related protein (MRP8) in calf lens. The relatively abundant expression of MIF in lens may have clinical significance, with the possibility of involvement in ocular inflammations that may follow damage to the lens.     

Disulfide bond mutations in follicle-stimulating hormone result in uncoupling of biological activity from intracellular behavior

The crystal structure of human CG reveals that each subunit is a member of the superfamily of cystine-knot growth factors. Although the distribution of the cysteine residues in all the beta-subunits is conserved, the conformation of the human FSH dimer differs from that of the CG/LH dimers. This suggests that the function of the cystine bonded loops in the human FSHbeta-subunit may differ from that in the CGbeta-subunit. To address this issue, we deleted two disulfide bonds in the FSHbeta domain: cys 20-104 and cys 28-82, which correspond to the disulfide bonds 26-110 and 34-88, respectively, in the CGbeta-subunit. The cys 26-110 bond is associated with the "seat-belt" region and cys 34-88 is a bond in the cystine knot. Coexpression of the wild-type alpha-subunit with the FSHbeta cysteine mutants in CHO cells revealed no detectable heterodimer. The FSHbeta mutants were then incorporated into a single chain where the beta-subunit is genetically fused to the alpha-subunit. In such a model, the rate-limiting subunit assembly step is by-passed and mutations that otherwise block heterodimer formation can be evaluated in terms of biological activity. Compared with the nonmutated single chain, the single-chain 28-82 mutant is secreted more slowly and its recovery is substantially reduced, whereas secretion and recovery of the 20-104 mutant was not significantly affected. The receptor binding affinity of the cys 28-82 mutant did not differ from wild-type and binding of the cys 20-104 mutant was decreased only 2-fold. The signal transduction data parallel the binding affinities, although the maximal accumulation of cAMP is less for the cys 20-104 mutant than that seen for cys 28-82 and nonmutated single-chains variants. These data support the hypothesis that the determinants for intracellular behavior and bioactivity of the gonadotropins are not the same, and that the cystine knot is a critical determinant for the formation of a stable, assembly-competent subunit. In addition, the data imply that the "seat-belt" conformation does not play a prominent role in the bioactivity of FSH.     

Synapse formation in response to estrogen in the medial amygdala developing in the eye.

Medial amygdaloid tissue, taken from female rats immediately after birth, was transplanted into the anterior chamber of the eye in adult ovariectomized host rats in order to elucidate the influence of estrogen on synapse formation without contribution of neural afferents. After injections of estradiol benzoate or oil vehicle to the hosts for 20 successive days, the grafts were processed for semiquantitative electron microscopic study to examine synaptic density in the neuropil. The number of synapses on dendritic shafts vs. dendritic spines was not significantly different in the control group. In contrast, in the grafts exposed to estrogen, shaft synapses occurred more frequently than spine synapses. Synaptic density on shafts was significantly greater in these grafts than that in the controls, although the density on spines did not differ between the two groups. These data show that estrogen affects the medial amygdaloid neurons themselves and specifically facilitates the formation of dendritic shaft synapses in oculo. Our previous report raises the possibility that the specific increase of shaft synapses induced by sex steroids is involved in the process of sexual differentiation of neuronal networks from the inherently feminine pattern to the masculine pattern in the medial amygdala. Therefore, the present findings may provide evidence that sexual differentiation triggered by sex steroids is accomplished by intrinsic factors in the neurons of the medial amygdala.     

Mutants in disulfide bond formation that disrupt flagellar assembly in Escherichia coli.

We report the isolation and characterization of Escherichia coli mutants (dsbB) that fail to assemble functional flagella unless cystine is present. Flagellar basal bodies obtained from these mutants are missing the L and P rings. This defect in assembly appears to result from an inability to form a disulfide bond in the P-ring protein (FlgI). Cystine suppresses this defect in dsbB strains. We also show that dsbA strains [Bardwell, J. C. A., McGovern, K. & Beckwith, J. (1991) Cell 67, 581-589] fail to assemble P rings, apparently from a similar failure in disulfide bond formation. However, cystine does not completely suppress this defect in dsbA strains. Thus, disulfide bond formation in FlgI is essential for assembly. DsbA likely puts in that bond directly, whereas the DsbB product(s) play a role in oxidizing DsbA, so that it can be active.     

Protein folding guides disulfide bond formation

The Anfinsen principle that the protein sequence uniquely determines its structure is based on experiments on oxidative refolding of a protein with disulfide bonds. The problem of how protein folding drives disulfide bond formation is poorly understood. Here, we have solved this long-standing problem by creating a general method for implementing the chemistry of disulfide bond formation and rupture in coarse-grained molecular simulations. As a case study, we investigate the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI). After confirming the experimental findings that the multiple routes to the folded state contain a network of states dominated by native disulfides, we show that the entropically unfavorable native single disulfide [14–38] between Cys₁₄ and Cys₃₈ forms only after polypeptide chain collapse and complete structuring of the central core of the protein containing an antiparallel β-sheet. Subsequent assembly, resulting in native two-disulfide bonds and the folded state, involves substantial unfolding of the protein and transient population of nonnative structures. The rate of [14–38] formation increases as the β-sheet stability increases. The flux to the native state, through a network of kinetically connected native-like intermediates, changes dramatically by altering the redox conditions. Disulfide bond formation between Cys residues not present in the native state are relevant only on the time scale of collapse of BPTI. The finding that formation of specific collapsed native-like structures guides efficient folding is applicable to a broad class of single-domain proteins, including enzyme-catalyzed disulfide proteins.The landmark discovery that the information to fold a protein is fully contained in the primary amino acid sequence was based on oxidative refolding experiments on disulfide bond formation in ribonuclease A (RNase A) (1, 2). Anfinsen showed that the initially unfolded protein, generated by reducing the disulfide (S–S) bonds in the native state of RNase A, folds reversibly under oxidizing conditions by correctly reforming the four native S–S bonds (among 105 possibilities) between the eight cysteine (Cys) residues. Besides being central to the enunciation of the principles of protein folding, many secretary proteins, whose misfolding is linked to a number of diseases, contain S–S bonds (3). Although biophysical aspects of such proteins are not as well studied as those without S–S bonds, understanding the link between conformational folding coupled to disulfide bond formation (4–7) is important and challenging both from a chemical and biophysical perspective (8).The formation of S–S bonds and their identities during folding can be monitored by quenching the oxidative process at various stages of the folding reaction (9). By arresting the reaction, it is possible to characterize the accumulated intermediates in terms of already formed S–S bonds (10). However, the relationship between protein folding and disulfide bond formation is nontrivial to establish because this requires separate reporters for disulfide bond formation and organization of the rest of the polypeptide chains. Even if the reaction can be arrested rapidly, the conformations of the intermediates are difficult to determine using experiments alone, although single molecule pulling experiments hold exceptional promise (7). Thus, well-calibrated computations are needed to decipher the precise relationship between conformational folding and S–S bond formation (11–14).Here, we investigate the coupling between conformational folding and disulfide bond formation by creating a novel way to mimic the effect of disulfide bond formation and rupture in coarse-grained (CG) molecular simulations, which have proven useful in a number of applications (15–18). As a case study, we use the 58-residue bovine pancreatic trypsin inhibitor (BPTI) with three S–S bonds in the native state to illustrate the key structural changes that occur during the folding reaction. The pioneering experiments of Creighton (9) seemed to indicate that nonnative disulfide species (19–22) are obligatory for productive folding to occur (for a thoughtful analysis, see ref. 23). Subsequently, using acid quench technique (by lowering pH, resulting in slowing down of the thiol disulfide exchange reaction) and a superior way of separating the intermediates Weissman and Kim (24) found that only native single and multiple disulfide bonds accumulate during the folding process. A plausible resolution of these contradictory findings was provided using theoretical studies (5) and simulations using lattice models (12) showing that nonnative intermediates are formed only on the time scale of the global collapse of the polypeptide chain. On longer times, only native species (S–S bonds found only in the folded state) dominate, as surmised by Weissman and Kim (24).The experimental studies could not resolve whether disulfide bond formation drives protein folding or vice versa, and has remained a major unsolved problem in protein folding. To solve this problem, we created a novel computational method to mimic disulfide bond formation and rupture within the context of a C_α representation of polypeptide chain by building on the demonstration by Scheraga and coworkers (6) that the formation or disruption of S–S bonds in these proteins can occur only if a few structurally important criteria (proximity of Cys residues, orientation, and accessibility of thiol groups to oxidative agents, see Figs. S1–S3) are met. We incorporated this physical insight in our model and simulated the oxidative folding of BPTI. Our results quantitatively capture the relative importance of all single and two disulfide intermediates that direct folding of BPTI. The initial rapid formation of single disulfide intermediates (in particular [14–38], an intermediate with disulfide bond between Cys₁₄ and Cys₃₈), occurs only after substantial compaction of BPTI and complete structuring of the central antiparallel β-sheet shown in Fig. 1A. Formation of two-disulfide intermediates and the species NSHSH ([5–55, 30–51]) that is poised to fold rapidly to the folded state N requires substantial unfolding of BPTI. Loop formation dictated by entropic considerations and forces that drive chain compaction place the Cys residues in proximity to enable S–S bond formation, thus directing BPTI to the folded state (5). Our work also provides a general framework to simulate oxidative folding of disulfide-containing proteins, and firmly establishes that early formation of specifically collapsed structures results in efficient folding of single domain proteins.Open in a separate windowFig. 1.(A) Ribbon diagram of the native structure of the 58 residue BPTI containing three disulfide bonds (marked in yellow) between residues Cys₅ and Cys₅₅ [5–55], Cys₁₄ and Cys₃₈ [14–38], and Cys₃₀ and Cys₅₁ [30–51], respectively. The antiparallel β-sheet is in red. (B) Simplified representation of the secondary tructure of BPTI and the three native disulfide bonds in BPTI. (C) Variables β_O and β_R mimicking the redox conditions. Small β_O (β_R) represent strongly oxidizing (reducing) condition. The star with β_O = 1.0 and β_R = 1.5 is used in most of the simulations. These values are a mixture of mildly oxidizing and reducing condition. (D) Distribution of fraction of native contacts obtained from high-temperature simulations.Open in a separate windowFig. S1.The mean values of the three structural parameters ?d_α?, 〈θαk〉, and ?n_α? obtained from equilibrium simulations at k_BT = 0.9? for BPTI containing with different combination of S–S bonds. Here, d_α is the distance between the two cysteines in native state, θαk (k=1,2) is the orientation angle defined in Fig. S2, and n_α is the number of residues within a spherical shell with radius R (Fig. S3). The label α = 1, 2, and 3 refer to the three disulfide bonds [5–55], [14–38], and [30–51], respectively. For example, for [5–55] (Left), these values are obtained from the simulations of BPTI mutants containing [5–55], N′, NSHSH, and N.Open in a separate windowFig. S3.The definition of the number of residues (n_α) within a spherical shell with radius R, drawn from O, the center of the α^th S–S bond.Open in a separate windowFig. S2.Definition of the orientation angle θ₁ and θ₂. The indices i − 1, i, i + 1 and j − 1, j, i + 1 are the two different peptide segments such that i and j are the two cysteines that can form a disulfide bond in the native state; θ₁ is the angle between the vector connecting the cysteine residues i and j (i < j) and the covalent bond connecting Cysⁱ and the neighboring Cαi−1. A similar definition holds for θ₂.     

Structural insights into peptide bond formation

The large ribosomal subunit catalyzes peptide bond formation and will do so by using small aminoacyl- and peptidyl-RNA fragments of tRNA. We have refined at 3-A resolution the structures of both A and P site substrate and product analogues, as well as an intermediate analogue, bound to the Haloarcula marismortui 50S ribosomal subunit. A P site substrate, CCA-Phe-caproic acid-biotin, binds equally to both sites, but in the presence of sparsomycin binds only to the P site. The CCA portions of these analogues are bound identically by either the A or P loop of the 23S rRNA. Combining the separate P and A site substrate complexes into one model reveals interactions that may occur when both are present simultaneously. The alpha-NH(2) group of an aminoacylated fragment in the A site forms one hydrogen bond with the N3 of A2486 (2451) and may form a second hydrogen bond either with the 2' OH of the A-76 ribose in the P site or with the 2' OH of A2486 (2451). These interactions position the alpha amino group adjacent to the carbonyl carbon of esterified P site substrate in an orientation suitable for a nucleophilic attack.     

Disulfide bond influence on protein structural dynamics probed with 2D-IR vibrational echo spectroscopy

Intramolecular disulfide bonds are understood to play a role in regulating protein stability and activity. Because disulfide bonds covalently link different components of a protein, they influence protein structure. However, the effects of disulfide bonds on fast (subpicosecond to approximately 100 ps) protein equilibrium structural fluctuations have not been characterized experimentally. Here, ultrafast 2D-IR vibrational echo spectroscopy is used to examine the constraints an intramolecular disulfide bond places on the structural fluctuations of the protein neuroglobin (Ngb). Ngb is a globin family protein found in vertebrate brains that binds oxygen reversibly. Like myoglobin (Mb), Ngb has the classical globin fold and key residues around the heme are conserved. Furthermore, the heme-ligated CO vibrational spectra of Mb (Mb-CO) and Ngb (Ngb-CO) are virtually identical. However, in contrast to Mb, human Ngb has an intramolecular disulfide bond that affects its oxygen affinity and protein stability. By using 2D-IR vibrational echo spectroscopy, we investigated the equilibrium protein dynamics of Ngb-CO by observing the CO spectral diffusion (time dependence of the 2D-IR line shapes) with and without the disulfide bond. Despite the similarity of the linear FTIR spectra of Ngb-CO with and without the disulfide bond, 2D-IR measurements reveal that the equilibrium sampling of different protein configurations is accelerated by disruption of the disulfide bond. The observations indicate that the intramolecular disulfide bond in Ngb acts as an inhibitor of fast protein dynamics even though eliminating it does not produce significant conformational change in the protein's structure.     

Enthalpy of hydrogen bond formation in a protein-ligand binding reaction.

Parallel measurements of the thermodynamics(free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding toFK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effortto probe the energetic contributions of single protein-ligand hydrogen bondsformed in the binding reactions. Changing tyrosine-82 to phenylalanine inFKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12complexes with tacrolimus or rapamycin and leads to a large apparent enthalpicstabilization of binding in both H2O and D2O. High-resolution crystallographicanalysis reveals that two water molecules bound to the tyrosine-82 hydroxylgroup in unliganded FKBP-12 are displaced upon formation of the protein-ligandcomplexes. A thermodynamic analysis is presented that suggests that the removalof polar atoms from water contributes a highly unfavorable enthalpy change tothe formation of C=O...HO hydrogen bonds as they occur in the processes ofprotein folding and ligand binding. Despite the less favorable enthalpy change,the entropic advantage of displacing two water molecules upon binding leads to aslightly more favorable free-energy change of binding in the reactions withwild-type FKBP-12.     

Phase separation in lens cytoplasm is genetically linked to cataract formation in the Philly mouse.

The variation of the phase-separation temperature, Tc, in lenses was studied during the postnatal development of three genetically different mouse strains: Swiss-Webster, Philly, and the (Swiss-Webster x Philly)F1 hybrid. The general behavior of Tc during early postnatal development has two stages: in stage I, Tc increased to a maximum and then, in stage II, Tc decreased. Philly mice are a strain that develops hereditary cataracts about 36 days following birth. In F1 hybrids of Philly and Swiss-Webster mice, cataracts appeared about 49 days following birth, approximately equal to 13 days later in development than in the Philly mice. In the Philly and hybrid mice, stage I and stage II were followed by stage III in which Tc reached a minimum value and then increased toward body temperature. The values of Tc at birth, the slope of the increase during stage I, and the maximum Tc were characteristic for each mouse strain. These results establish that the behavior of the temperature of the phase separation Tc in mouse lens is linked to the genetic strain of the mice and that the value of Tc at birth is an early indicator of lenses that will develop cataracts and lenses that will develop normally.     

Observation of liquid-liquid phase separation for eye lens gammaS-crystallin

gammaS-crystallin (gammaS) is an important human and bovine eye lens protein involved in maintaining the transparency of the eye. By adding small amounts of polyethylene glycol (PEG) to the binary aqueous bovine gammaS solutions, we have observed liquid-liquid phase separation (LLPS) at -8 degrees C and revealed that, in the binary gammaS-water system, this phase transition would occur at -28 degrees C. We have measured both the effect of PEG concentration on the LLPS temperature and proteinPEG partitioning between the two liquid coexisting phases. We use our measurements of proteinPEG partitioning to determine the nature and the magnitude of the gammaS-PEG interactions and to quantitatively assess the effectiveness of PEG as a crystallizing agent for gammaS. We use our measurements of LLPS temperature as a function of protein and PEG concentration to successfully determine the location of the critical point for the binary gammaS-water system. This phase transition cannot be observed in the absence of PEG because it is inaccessible due to the freezing of the system. Our findings indicate that the effective interactions between gammaS molecules in the binary gammaS-water system are attractive. We compare the magnitude of the attraction found for gammaS with the results obtained for the other gamma-crystallins for which the critical temperature is located above the freezing point of the system. This work suggests that PEG can be used to reveal the existence of LLPS for a much wider range of binary protein-water systems than known previously.     

The transition state for formation of the peptide bond in the ribosome

Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3' end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs. The calculated TS activation energy, E(a), is 35.5 kcal (1 kcal = 4.18 kJ)/mol, and the increase in hydrogen bonding between the rotating A-site tRNA and ribosome nucleotides as the TS forms appears to stabilize it to a value qualitatively estimated to be approximately 18 kcal/mol. The optimized geometry corresponds to a structure in which the peptide bond is being formed as other bonds are being broken, in such a manner as to release the P-site tRNA so that it may exit as a free molecule and be replaced by the translocating A-site tRNA. At TS formation the 2' OH group of the P-site tRNA A76 forms a hydrogen bond with the oxygen atom of the carboxyl group of the amino acid attached to the A-site tRNA, which may be indicative of its catalytic role, consistent with recent biochemical experiments.