Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

Thermal,Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation

Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol⁻¹ and 14.90 Kcal mol⁻¹ for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔC_p) is 3.42 Kcal mol⁻¹ K⁻¹ for dimer. The small ΔC_p for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.     

Heterogeneity of Equilibrium Molten Globule State of Cytochrome c Induced by Weak Salt Denaturants under Physiological Condition

While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO₄) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε ₄₀₀) and CD ([θ]₄₀₉), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO₄ and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO₄ and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.     

Probing membrane protein unfolding with pulse proteolysis

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A₅₆₀). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (C_m) shows excellent agreement with that determined by A₅₆₀. The C_m values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A₅₆₀. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.     

Predicting loop–helix tertiary structural contacts in RNA pseudoknots

Tertiary interactions between loops and helical stems play critical roles in the biological function of many RNA pseudoknots. However, quantitative predictions for RNA tertiary interactions remain elusive. Here we report a statistical mechanical model for the prediction of noncanonical loop–stem base-pairing interactions in RNA pseudoknots. Central to the model is the evaluation of the conformational entropy for the pseudoknotted folds with defined loop–stem tertiary structural contacts. We develop an RNA virtual bond-based conformational model (Vfold model), which permits a rigorous computation of the conformational entropy for a given fold that contains loop–stem tertiary contacts. With the entropy parameters predicted from the Vfold model and the energy parameters for the tertiary contacts as inserted parameters, we can then predict the RNA folding thermodynamics, from which we can extract the tertiary contact thermodynamic parameters from theory–experimental comparisons. These comparisons reveal a contact enthalpy (ΔH) of −14 kcal/mol and a contact entropy (ΔS) of −38 cal/mol/K for a protonated C⁺•(G–C) base triple at pH 7.0, and (ΔH = −7 kcal/mol, ΔS = −19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory–experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop–stem tertiary contacts from the nucleotide sequence alone.     

Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of ΔC p of protein unfolding

Cold and heat denaturation of the double mutant Arg 3→Glu/Leu 66→Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D ¹H NMR spectroscopy in the temperature range from −12°C in supercooled water up to +70°C. The fraction of unfolded protein, f _u, was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be −11°C and 39°C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, ΔC _p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.     

Ligand-Induced Changes of the Apparent Transition-State Position in?Mechanical Protein Unfolding

Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx^‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx^‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx^‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.     

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔH_ITC–Δ(TΔS) compensation, negative ΔΔV and a net release of counterions. The opposite signs of the differential enthalpy–entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the ‘modified’ duplex.     

Thermal,Chemical and pH Induced Denaturation of a Multimeric β-Galactosidase Reveals Multiple Unfolding Pathways

Background

In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea β-galactosidase (CpGAL), as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH.

Methodology/Principal Findings

CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to α+β class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50°C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (ΔG₀) and unfolding constant (K_obs) were also calculated for chemically denatured CpGAL.

Significance

The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well.     

Monomeric Banana Lectin at Acidic pH Overrules Conformational Stability of Its Native Dimeric Form

Banana lectin (BL) is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS) binding, size exclusion chromatography (SEC) and dynamic light scattering (DLS). During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml) at pH 2.0 while single peak (61.45 ml) at pH 7.4. The hydrodynamic radii (R _h) of native BL was 2.9 nm while at pH 2.0 two species were found with R _h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high C_m and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.     

Bacillus subtilis polynucleotide phosphorylase 3′-to-5′ DNase activity is involved in DNA repair

In the presence of Mn²⁺, an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3′ → 5′ polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn²⁺ and low-level inorganic phosphate (P_i), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg²⁺ or high-level P_i. In contrast, the RNase activity of PNPase requires Mg²⁺ and P_i, suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (ΔpnpA) is not epistatic with ΔrecA, but is epistatic with ΔrecN and Δku, which by themselves are non-epistatic. The addA5, ΔrecO, ΔrecQ (ΔrecJ), ΔrecU and ΔrecG mutations (representative of different epistatic groups), in the context of ΔpnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways.     

Competitive Model on Denaturant-Mediated Protein Unfolding

A denaturant-mediated protein unfolding model, which is different from already existing ones based on the assumption that denaturant competes for water molecules to interact and thus reduces water–protein interactions, which leads to unfolding phenomenon, has been developed with a detailed mathematical justification. Theoretical results suggested that the parameter (m_u) obtained from the usual linear extrapolation model must be a linear function of the number of bound water molecules (n) on protein with a zero intercept. However, application of this theory to a set of proteins for which m_u values for urea denaturation are already known showed that m_u was a linear function of n but with a nonzero intercept. Finally this nonzero intercept was attributed to binding of denaturant to protein at n = 0. Detailed investigation of this factor showed that average equilibrium constant for binding of urea with aromatic side chains (generally nonpolar side chains) was k_b ≈ 0.65 ± 0.45 mol⁻¹, which agreed well with earlier experimental estimations, and also suggested that an integrated approach was necessary to avoid discrepancy in ΔG^H₂O estimated from different models.     

Measuring “Unmeasurable” Folding Kinetics of Proteins by Single-Molecule Force Spectroscopy

Folding and unfolding are fundamental biological processes in cell and are important for the biological functions of proteins. Characterizing the folding and unfolding kinetics of proteins is important for understanding the energetic landscape leading to the active native conformations of these molecules. However, the thermal or chemical-induced unfolding of many proteins is irreversible in vitro, precluding characterization of the folding kinetics of such proteins, just as it is impossible to “un-boil” an egg. Irreversible unfolding often manifests as irreversible aggregation of unfolded polypeptide chains, which typically occurs between denatured protein molecules in response to the exposure of hydrophobic residues to solvent. An example of such a protein where thermal denaturation results in irreversible aggregation is the β-1,4 endoxylanase from Bacillus circulans (BCX). Here, we report the use of single-molecule atomic force microscopy to directly measure the folding kinetics of BCX in vitro. By mechanically unfolding BCX, we essentially allowed only one unfolded molecule to exist in solution at a given time, effectively eliminating the possibility for aggregation. We found that BCX can readily refold back to the native state, allowing us to measure its folding kinetics for the first time. Our results demonstrate that single-molecule force-spectroscopy-based methods can adequately tackle the challenge of “un-boiling eggs”, providing a general methodology to characterize the folding kinetics of many proteins that suffer from irreversible denaturation and thus cannot be characterized using traditional equilibrium methodologies.     

Cooperative Unfolding of Residual Structure in Heat Denatured Proteins by Urea and Guanidinium Chloride

The denatured states of proteins have always attracted our attention due to the fact that the denatured state is the only experimentally achievable state of a protein, which can be taken as initial reference state for considering the in vitro folding and defining the native protein stability. It is known that heat and guanidinium chloride (GdmCl) give structurally different states of RNase-A, lysozyme, α-chymotrypsinogen A and α-lactalbumin. On the contrary, differential scanning calorimetric (DSC) and isothermal titration calorimetric measurements, reported in the literature, led to the conclusion that heat denatured and GdmCl denatured states are thermodynamically and structurally identical. In order to resolve this controversy, we have measured changes in the far-UV CD (circular dichroism) of these heat-denatured proteins on the addition of different concentrations of GdmCl. The observed sigmoidal curve of each protein was analyzed for Gibbs free energy change in the absence of the denaturant (ΔG ⁰ _X→D) associated with the process heat denatured (X) state ↔ GdmCl denatured (D) state. To confirm that this thermodynamic property represents the property of the protein alone and is not a manifestation of salvation effect, we measured urea-induced denaturation curves of these heat denatured proteins under the same experimental condition in which GdmCl-induced denaturation was carried out. In this paper we report that (a) heat denatured proteins contain secondary structure, and GdmCl (or urea) induces a cooperative transition between X and D states, (b) for each protein at a given pH and temperature, thermodynamic cycle connects quantities, ΔG ⁰ _N→X (native (N) state ↔ X state), ΔG ⁰ _X→D and ΔG ⁰ _N→D (N state ↔ D state), and (c) there is not a good enthalpy difference between X and D states, which is the reason for the absence of endothermic peak in DSC scan for the transition, X state ↔ D state.     

The Role of Allosteric Coupling on Thermal Activation of Thermo-TRP Channels

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔH^o) and entropy (ΔS^o) upon channel gating. The magnitude, sign, and temperature dependence of ΔH^o and ΔS^o, the last given by an associated change in heat capacity (ΔC_p), can determine a channel’s temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔC_p makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔH^o and ΔS^o, including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔH^o/ΔS^o ratio associated with them, can also determine a channel’s temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔC_p-independent manner.     

Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n-Alkane-Assimilating Yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C₁₀ to C₁₃). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C₄ substrate butyrate.     

Yeast telomerase appears to frequently copy the entire template in vivo

Telomeres derived from the same formation event in wild type strains of Saccharomyces cerevisiae possess the same, precise TG_1–3 sequence for the most internal ~100 bp of the 250–350 bp TG_1–3 repeats. The conservation of this internal domain is thought to reflect the fact that telomere lengthening and shortening, and thus alteration of the precise TG_1–3 sequence, is confined to the terminal region of the telomere. The internal domains of telomeres from yku70Δ and tel1Δ mutants, whose entire telomeres are only ~100 bp, were examined by analyzing 5.1 kb of cloned TG_1–3 sequences from telomeres formed during transformation of wild type, yku70Δ and tel1Δ cells. The internal domains were 97–137 bp in wild type cells, 27–36 bp in yku70Δ cells and 7–9 bp in tel1Δ cells. These data suggest that the majority of the tel1Δ cell TG_1–3 repeats may be resynthesized during shortening and lengthening reactions while a portion of the yku70Δ cell telomeres are protected. TG_1–3 sequences are synthesized by telomerase repeatedly copying an internal RNA template, which introduces a sequence bias into TG_1–3 repeats. Analysis of in vivo-derived telomeres revealed that of the many possible high affinity binding sites for the telomere protein Rap1p in TG_1–3 repeats, only those consistent with telomere hybridization to the ACACAC in the 3′-region of the telomerase RNA template followed by copying of most of the template were present. Copies of the telomerase RNA template made up 40–60% of the TG_1–3 sequences from each strain and could be found in long, tandem repeats. The data suggest that in vivo yeast telomerase frequently allows telomeres to hybridize to the 3′-region of RNA template and copy most of it prior to dissociation, or that in vivo telomere processing events result in the production of TG_1–3 sequences that mimic this process.     

An Element of Determinism in a Stochastic Flagellar Motor Switch

Marine bacterium Vibrio alginolyticus uses a single polar flagellum to navigate in an aqueous environment. Similar to Escherichia coli cells, the polar flagellar motor has two states; when the motor is counter-clockwise, the cell swims forward and when the motor is clockwise, the cell swims backward. V. alginolyticus also incorporates a direction randomization step at the start of the forward swimming interval by flicking its flagellum. To gain an understanding on how the polar flagellar motor switch is regulated, distributions of the forward Δ_f and backward Δ_b intervals are investigated herein. We found that the steady-state probability density functions, P(Δ_f) and P(Δ_b), of freely swimming bacteria are strongly peaked at a finite time, suggesting that the motor switch is not Poissonian. The short-time inhibition is sufficiently strong and long lasting, i.e., several hundred milliseconds for both intervals, which is readily observed and characterized. Treating motor reversal dynamics as a first-passage problem, which results from conformation fluctuations of the motor switch, we calculated P(Δ_f) and P(Δ_b) and found good agreement with the measurements.     

Carbon isotope discrimination varies genetically in c(4) species

Carbon-isotope discrimination (Δ) is used to distinguish between different photosynthetic pathways. It has also been shown that variation in Δ occurs among varieties of C₃ species, but not as yet, in C₄ species. We now report that Δ also varies among genotypes of sorghum (Sorghum bicolor Moench), a C₄ species. The discrimination in leaves of field-grown plants of 12 diverse genotypes of sorghum was measured and compared with their grain yields. Discrimination varied significantly among genotypes, and there was a significant negative correlation between grain yield and Δ. The variation in Δ may be caused by genetic differences in either leakiness of the bundle-sheath cells or by differences in the ratio of assimilation rate to stomatal conductance. At the leaf level, the former should be related to light-use efficiency of carbon fixation and the latter should be related to transpiration efficiency. Both could relate to the yield of the crop.