Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the λ Cro-V55C repressor protein

A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the λ Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel β-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20–95°C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created in three (closely related) steps. A first step entails the unfolding of the short N-terminal β-strand, followed by the unfolding of the α-helices in a second step, and the third step comprises the reorganization of the remaining β-sheet and of some unordered segments in the protein. The complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two β-strands at the dimer interface.

Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria

A negative feedback control of kaiC expression by KaiC protein has been proposed to generate a basic oscillation of the circadian clock in the cyanobacterium Synechococcus sp. PCC 7942. KaiC has two P loops or Walker's motif As, that are potential ATP-/GTP-binding motifs and DXXG motifs conserved in various GTP-binding proteins. Herein, we demonstrate that in vitro KaiC binds ATP and, with lower affinity, GTP. Point mutation by site-directed mutagenesis of P loop 1 completely nullified the circadian rhythm of kaiBC expression and markedly reduced ATP-binding activity. Moreover, KaiC can be autophosphorylated in vitro. These results suggest that the nucleotide-binding activity of KaiC plays important roles in the generation of circadian oscillation in cyanobacteria.

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423—Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy

We report the study of the dynamics of the unbinding process under a force load f of adsorbed proteins (fibrinogen) on a solid surface (hydrophilic silica) by means of atomic force microscopy spectroscopy. By varying the loading rate rf, defined by f = rf t, t being the time, we find that, as for specific interactions, the mean rupture force increases with rf. This unbinding process is analyzed in the framework of the widely used Bell model. The typical dissociation rate at zero force entering in the model lies between 0.02 and 0.6 s−1. Each measured rupture is characterized by a force f0, which appears to be quantized in integer multiples of 180–200 pN.

Initiation process of earthquakes and its implications for seismic hazard reduction strategy.

For the average citizen and the public, "earthquake prediction" means "short-term prediction," a prediction of a specific earthquake on a relatively short time scale. Such prediction must specify the time, place, and magnitude of the earthquake in question with sufficiently high reliability. For this type of prediction, one must rely on some short-term precursors. Examinations of strain changes just before large earthquakes suggest that consistent detection of such precursory strain changes cannot be expected. Other precursory phenomena such as foreshocks and nonseismological anomalies do not occur consistently either. Thus, reliable short-term prediction would be very difficult. Although short-term predictions with large uncertainties could be useful for some areas if their social and economic environments can tolerate false alarms, such predictions would be impractical for most modern industrialized cities. A strategy for effective seismic hazard reduction is to take full advantage of the recent technical advancements in seismology, computers, and communication. In highly industrialized communities, rapid earthquake information is critically important for emergency services agencies, utilities, communications, financial companies, and media to make quick reports and damage estimates and to determine where emergency response is most needed. Long-term forecast, or prognosis, of earthquakes is important for development of realistic building codes, retrofitting existing structures, and land-use planning, but the distinction between short-term and long-term predictions needs to be clearly communicated to the public to avoid misunderstanding.

Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.

During fertilization in marine invertebrates, fusion between sperm and egg cell membranes occurs at the tip of the sperm acrosomal process. In abalone sperm the acrosomal process is coated with an 18-kDa protein. In situ, this protein has no effect on the egg vitelline envelope, but in vitro it is a potent fusagen of liposomes. Thus, the 18-kDa protein may mediate membrane fusion between the gametes, a step in gamete recognition known to restrict heterospecific fertilization in other species. The cDNA and deduced amino acid sequences of the 18-kDa protein were determined for five species of California abalone. The deduced amino acid sequences exhibit extraordinary divergence; the percent identity varies from 27% to 87%. Analysis of nucleotide substitution shows extremely high frequencies of amino acid-altering substitution compared to silent substitution, demonstrating that positive Darwinian selection promotes the divergence of this protein. However, amino acid replacement is conservative with respect to size and polarity of residue. The data support the developing idea that in free-spawning marine invertebrates, the proteins mediating fertilization may be subjected to intense, and as yet unknown, selective forces. The extraordinary divergence of fertilization proteins may be related to the establishment of barriers to heterospecific fertilization.

Kinetics of hydrogen bond breakage in the process of unfolding of ribonuclease A measured by pulsed hydrogen exchange.

A sensitive test for kinetic unfolding intermediates in ribonuclease A (EC 3.1.27.5) is performed under conditions where the enzyme unfolds slowly (10 degrees C, pH 8.0, 4.5 M guanidinium chloride). Exchange of peptide NH protons (2H-1H) is used to monitor structural opening of individual hydrogen bonds during unfolding, and kinetic models are developed for hydrogen exchange during the process of protein unfolding. The analysis indicates that the kinetic process of unfolding can be monitored by EX1 exchange (limited by the rate of opening) for ribonuclease A in these conditions. Of the 49 protons whose unfolding/exchange kinetics was measured, 47 have known hydrogen bond acceptor groups. To test whether exchange during unfolding follows the EX2 (base-catalyzed) or the EX1 (uncatalyzed) mechanism, unfolding/exchange was measured both at pH 8.0 and at pH 9.0. A few faster-exchanging protons were found that undergo exchange by both EX1 and EX2 processes, but the 43 slower-exchanging protons at pH 8 undergo exchange only by the EX1 mechanism, and they have closely similar rates. Thus, it is likely that all 49 protons undergo EX1 exchange at the same rate. The results indicate that a single rate-limiting step in unfolding breaks the entire network of peptide hydrogen bonds and causes the overall unfolding of ribonuclease A. The additional exchange observed for some protons that follows the EX2 mechanism probably results from equilibrium unfolding intermediates and will be discussed elsewhere.

Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20–30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-β (TGFβ); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFβ. A strong correlation between capacity to maintain growth in the presence of TGFβ and expression of telomerase activity was observed. We have used the term “conversion” to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFβ and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.

Redox-dependent activation of CO dehydrogenase from Rhodospirillum rubrum

Studies of initial activities of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum show that CODH is mostly inactive at redox potentials higher than −300 mV. Initial activities measured at a wide range of redox potentials (0–500 mV) fit a function corresponding to the Nernst equation with a midpoint potential of −316 mV. Previously, extensive EPR studies of CODH have suggested that CODH has three distinct redox states: (i) a spin-coupled state at −60 to −300 mV that gives rise to an EPR signal termed Cred1; (ii) uncoupled states at <−320 mV in the absence of CO2 referred to as Cunc; and (iii) another spin-coupled state at <−320 mV in the presence of CO2 that gives rise to an EPR signal termed Cred2B. Because there is no initial CODH activity at potentials that give rise to Cred1, the state (Cred1) is not involved in the catalytic mechanism of this enzyme. At potentials more positive than −380 mV, CODH recovers its full activity over time when incubated with CO. This reductant-dependent conversion of CODH from an inactive to an active form is referred to hereafter as “autocatalysis.” Analyses of the autocatalytic activation process of CODH suggest that the autocatalysis is initiated by a small fraction of activated CODH; the small fraction of active CODH catalyzes CO oxidation and consequently lowers the redox potential of the assay system. This process is accelerated with time because of accumulation of the active enzyme.

Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.

Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex.