Intermolecular protein interactions in solutions of calf lens alpha-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles.

From analyses of the magnetic field dependence of 1/T1 (NMRD profiles) of water protons in solutions of calf lens alpha-crystallin at several concentrations, we find two regimes of solute behavior in both cortical and nuclear preparations. Below approximately 15% vol/vol protein concentration, the solute molecules appear as compact globular proteins of approximately 1,350 (cortical) and approximately 1,700 (nuclear) kD. At higher concentrations, the effective solute particle size increases, reversibly, as evidenced by the appearance of spectra-like 14N peaks in the NMRD profiles and a change in the field and temperature dependence of 1/T1. At these higher concentrations, the profiles are very similar to those of calf gamma II-crystallin, a crystallin that undergoes an analogous transition near approximately 15% protein (Koenig, S. H., C.F. Beaulieu, R. D. Brown III, and M. Spiller, 1990. Biophys. J. 57:461-469). By comparison with recent analyses of NMRD results for solutions of immobilized proteins as models for the transition from protein solutions to tissue (Koenig, S. H., and R. D. Brown III. 1991. Prog. NMR Spectr. 22:487-567), we argue that alpha-crystallin solute behaves as aggregates approximately greater than 50,000 kD as protein concentration is progressively increased above 15%. Finally, the concentration dependence of the NMRD profiles of alpha- and gamma II-crystallin can readily explain recent osmotic pressure data, in particular the intersection of the respective pressure curves at approximately 23% vol/vol (Vérétout, F., and A. Tardieu. 1989. Eur. Biophys. J. 17:61-68).     

Oligomerization and conformation change in solutions of calf lens gamma II-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles.

From analyses of the magnetic field dependence of 1/T1 (nuclear magnetic relaxation dispersion [NMRD] profiles) of water protons in solutions of highly purified calf lens gamma II-crystallin, we find that monomers form oligomers at relatively low concentrations, which increase in size with increasing concentration and decreasing temperature. At approximately 16% by volume and -4 degrees C, the mean oligomeric molecular weight is approximately 120-fold greater than the monomeric value of 20 kD. Below this concentration, there is no indication of any substantive change in conformation of the monomeric subunits. At higher concentrations, the tertiary structure of the monomer appears to reconfigure rather abruptly, but reversibly, as evidenced by the appearance of spectra-like 14N peaks in the NMRD profiles. The magnitudes of these peaks, known to arise from cross-relaxation of water protons through access to amide (NH) moieties of the protein backbone, indicate that the high concentration conformation is not compact, but open and extended in a manner that allows enhanced interaction with solvent. The data are analogous to those found for homogenates of calf and chicken lens (Beaulieu, C. F., J. I. Clark, R. D. Brown III, M. Spiller, and S. H. Koenig. 1988. Magn. Reson. Med. 8:47-57; Beaulieu, C. F., R. D. Brown III, J. I. Clark, M. Spiller, and S. H. Koenig. 1989. Magn. Reson. Med. 10:62-72). This unusually large dependence of oligomeric size and conformation on concentration in the physiological range is suggested as the mechanism by which osmotic equilibrium is maintained, at minimal metabolic expense, in the presence of large gradients of protein concentration in the lens in vivo (cf Vérétout and Tardieu, 1989. Eur. Biophys. J. 17:61-68). Finally, the results of the NMRD data provide a ready explanation of the low temperature phase transition, and "cold-cataract" separation of phases, observed in gamma II-crystallin solutions; we suggest that the phases that separate are the two major conformers detected by NMRD.     

Alpha-crystallin exists in a non-spherical form. A study on the rotational properties of native and reconstituted alpha-crystallin.

Native alpha-crystallin, obtained from the cortex of calf lenses with FPLC (Pharmacia) was characterized by means of transient-electric-birefringence measurements and ultraviolet linear-dichroism spectroscopy. These techniques were also performed on 6-M-urea-dissociated and reconstituted alpha-crystallin. Transient-electric-birefringence measurements offer the possibility to characterize the often observed, but usually neglected, non-spherical occurrences of alpha-crystallin in more detail. Although not distinguishable with size-exclusion chromatography, we could identify at least two different classes of both native and reconstituted alpha-crystallin, from which at least one consists of non-spherical molecules. The results are compared with those obtained with electron microscopy using different staining methods. From the three independent techniques used we find evidence that a fraction of the alpha-crystallin exists in a more extended quaternary structure. The results are difficult to explain with a concentric three-layer model for alpha-crystallin as proposed by Tardieu et al. [Tardieu, A., Laporte, D., Licinio, P., Krop, B. & Delaye, M. (1986) J. Mol. Biol. 192, 711-724].     

Direct determination of crystallographic phases for diffraction data from lipid bilayers. I. Reliability and phase refinement.

Direct analysis of lipid lamellar packing based on the probabilistic estimate of sigma 1- and sigma 2-triplet phase invariants is evaluated here for a large variety of bilayer structures than examined in an original study of this problem (Dorset, D.L., 1990. Biophys. J. 58:1077-1087). Using x-ray crystal structures of five phospholipids, three glycerides and two cerebrosides, lamellar diffraction data were generated at the approximately 3 A resolution often found experimentally from oriented multilayers. For structures where no significant density occurs at the unit cell origin, the ab initio phase determination is successful for six of the ten structures. A seventh structure can be solved if a limited set of sigma 2-triples are used to determine the initial phase set based on the hierarchy of the A2 values. Bilayers, e.g., with solvent at the origin, can be analyzed if a modified criterion for accepting phase estimates for sigma 1-triples is used, as suggested by the distribution of normalized structure factors and the number of probable single-valued phase domains. In all cases, partial phase determinations can be refined effectively by density modification ("flattening") of the hydrocarbon region in real space. A figure of merit suggested by Luzzati et al. (Luzzati, V., A. Tardieu, and D. Taupin. 1972. J. Mol. Biol. 64:269-286) used to evaluate the success of such refinement can be supplemented by an evaluation of density smoothness, which can also detect the presence of near structure homomorphs not identified by the former test for density flatness.     

Simultaneous measurement of electric birefringence and dichroism. A study on photosystem 1 particles.

We have developed a straightforward method to separate linear-dichroism and birefringence contributions to electric-field induced signals in a conventional birefringence setup. The method requires the measurement of electric birefringence for three different angular positions of the analyzer. It is demonstrated that the presence of linear dichroism can significantly influence the measured signals and lead to completely erroneous calculations of the birefringence signal and field-free decay times if its contribution is not taken into account. The new method is used to determine electric birefringence and linear dichroism of trimeric Photosystem 1 complexes from the cyanobacterium Synechocystis PCC 6803 in the detergents n-dodecyl-beta-D-maltoside and n-octyl-beta-D-glucoside. It is concluded that the orientation of the particles in the field is predominantly caused by a permanent electric dipole moment that is directed parallel to the symmetry axis of the particles. Comparison of the decay times obtained with dodecylmaltoside and octylglucoside supports a model in which the thickness of the disc-like complexes remains similar (7-8 nm) upon replacing dodecylmaltoside by octylglucoside, whereas the diameter increases from 14.4 +/- 0.2 to 16.6 +/- 0.2 nm because of an increased thickness of the detergent layer. This change in diameter is in good agreement with electron-microscopy results on Photosystem 2 complexes in dodecylmaltoside and octylglucoside (Dekker, J. P., E. J. Boekema, H. T. Witt, and M. Rögner. 1988. Biochim. Biophys. Acta 936:307-318). The value of approximately 16.6 nm for the diameter of Photosystem 1 trimers in dodecylmaltoside is in good agreement with recent results obtained from electron microscopy in combination with extensive image analysis (Kruip, J., E. J. Boekema, D. Bald, A. F. Boonstra, and M. Rögner. 1993. J. Biol. Chem. 268:23353-23360).     

MITOCHONDRIAL PROTEIN SYNTHESIS IN HELA CELLS

HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1½–2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a–a₃ and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.     

Micropipet-based pico force transducer: in depth analysis and experimental verification.

Measurements of forces in the piconewton range are very important for the study of molecular adhesion and mechanics. Recently, a micropipet-based force transducer for this type of experiment was presented (E. Evans, K. Ritchie, and R. Merkel, 1995, Biophys. J., 68:2580-2587). In the present article we give a detailed mechanical analysis of this transducer, including nonlinear effects. An analytical expression for the transducer stiffness at small elongations is given. Using magnetic tweezers (F. Ziemann, J. Rädler, and E. Sackmann, 1994, Biophys. J., 66:2210-2216), we were able to determine the force displacement relation of this transducer experimentally. Forces from approximately 10 pN to 500 pN were applied. Theoretical predictions and experimental results coincide remarkably well.     

Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications.

The genes (selC) coding for the selenocysteine-inserting tRNA species (tRNA(Sec)) from Clostridium thermoaceticum and Desulfomicrobium baculatum were cloned and sequenced. Although they differ in numerous positions from the sequence of the Escherichia coli selC gene, they were able to complement the selC lesion of an E. coli mutant and to promote selenoprotein formation in the heterologous host. The tRNA(Sec) species from both organisms possess all of the unique primary, secondary, and tertiary structural features exhibited by E. coli tRNA(Sec) (C. Baron, E. Westhof, A. Böck, and R. Giegé, J. Mol. Biol. 231:274-292, 1993). The structural and functional properties of the tRNA(Sec) species from prokaryotes analyzed thus far support the notion that tRNA(Sec) may be an evolutionarily conserved structure whose function in the primordial genetic code was to decode UGA with selenocysteine.     

Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase/oxygenase.

Active-site His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3-hydroxyl of bound substrate or reaction-intermediate analogue (CABP), water molecules, and ligands for the activator metal-ion (Andersson I, 1996, J Mol Biol 259:160-174; Taylor TC, Andersson I, 1997, J Mol Biol 265:432-444). To test structure-based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild-type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (approximately 10(3)-fold and > 10(5)-fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10-fold reduced CO2/O2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward-processing of carboxylated intermediate.     

Analysis of the high- and low-spin Soret bands of horse-heart metmyoglobin complexes

Interest in the thermodynamics of the iron-binding site in hemoproteins has increased in recent years due to refinements in x-ray crystallographic studies of hemoproteins [see Deathage, J. F., Lee, R. S., Anderson, C. M. & Moffat, K. (1976) J. Mol. Biol. 104 , 687–706; Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976) J. Mol. Biol. 104 , 707–722; Deathage, J. F., Lee, R. S. & Moffat, K. (1976) J. Mol. Biol. 104 , 723–728; Ladner, R. C., Heidner, E. J. & Perutz, M. F. (1976) J. Mol. Biol. 114 , 385–414; Fermi, G. & Perutz, M. F. (1977) J. Mol. Biol. 114 , 421–431; Takano, T. (1977) J. Mol. Biol. 110 , 537–568 and 569–589], the synthesis and x-ray analysis of model heme compounds [see Scheidt, W. R. (1977) Acc. Chem. Res. 10 , 339–345; Kastner, M. E., Scheidt, W. R., Mashino, T. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 666–667; Mashiko, T., Kastner, M. E., Spartalian, K., Scheidt, W. R. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 6354–6362; Hill, H. A. O., Skite, P. P., Buchler, J. W., Luchr, H., Tonn, M., Gregson, A. K. & Pellizer, G. (1979) Chem. Commun. 4 , 151–152; and Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979) Biochemistry 18 , 3546–3556], and the numerous data on heme–protein interactions that account for the differences observed in ligand binding between the various species of animals. Numerous probes have been used and provide information about the structure and thermodynamics of the binding site, but no single probe can provide the complete picture [see Iizuka, T. & Yonetani, T. (1970) Adv. Biophys. 1 , 157–182; Smith, D. W. & Williams, R. J. P. (1970) Struct. Bond. 7 , 1–45; and Spiro, T. G. (1975) Biochim. Biophys. Acta 416 , 169–189].     

Crystal structure of activated tobacco rubisco complexed with the reaction-intermediate analogue 2-carboxy-arabinitol 1,5-bisphosphate.

The crystal structure of activated tobacco rubisco, complexed with the reaction-intermediate analogue 2-carboxy-arabinitol 1,5-bisphosphate (CABP) has been determined by molecular replacement, using the structure of activated spinach rubisco (Knight, S., Andersson, I., & Brändén, C.-I., 1990, J. Mol. Biol. 215, 113-160) as a model. The R-factor after refinement is 21.0% for 57,855 reflections between 9.0 and 2.7 A resolution. The local fourfold axis of the rubisco hexadecamer coincides with a crystallographic twofold axis. The result is that the asymmetric unit of the crystals contains half of the L8S8 complex (molecular mass 280 kDa in the asymmetric unit). The activated form of tobacco rubisco is very similar to the activated form of spinach rubisco. The root mean square difference is 0.4 A for 587 equivalent C alpha atoms. Analysis of mutations between tobacco and spinach rubisco revealed that the vast majority of mutations concerned exposed residues. Only 7 buried residues were found to be mutated versus 54 residues at or near the surface of the protein. The crystal structure suggests that the Cys 247-Cys 247 and Cys 449-Cys 459 pairs are linked via disulfide bridges. This pattern of disulfide links differ from the pattern of disulfide links observed in crystals of unactivated tobacco rubisco (Curmi, P.M.G., et al., 1992, J. Biol. Chem. 267, 16980-16989) and is similar to the pattern observed for activated spinach tobacco.     

Simian virus 40 replication pause sites map with the nucleosomes.

The locations of replication pause sites in the simian virus 40 minichromosome which were determined by sizing cloned fragments of nascent DNA (Zannis-Hadjopoulos et al., J. Mol. Biol. 165:599-607, 1983) were compared with the positions of simian virus 40 nucleosomes in the genome, as obtained by sequence-directed mapping (G. Mengeritsky and E. N. Trifonov, Nucleic Acids Res. 11:3833-3851, 1983; Mengeritsky and Trifonov, Cell Biophys. 6:1-8, 1984). Clear correlation between these two maps is demonstrated, suggesting that nucleosomes hinder propagation of the replication forks.     

AFM review study on pox viruses and living cells.

Single living cells were studied in growth medium by atomic force microscopy at a high--down to one image frame per second--imaging rate over time periods of many hours, stably producing hundreds of consecutive scans with a lateral resolution of approximately 30-40 nm. The cell was held by a micropipette mounted onto the scanner-piezo as shown in Häberle, W., J. K. H. Hörber, and G. Binnig. 1991. Force microscopy on living cells. J. Vac. Sci. Technol. B9:1210-0000. To initiate specific processes on the cell surface the cells had been infected with pox viruses as reported earlier and, most likely, the liberation of a progeny virion by the still-living cell was observed, hence confirming and supporting earlier results (Häberle, W., J. K. H. Hörber, F. Ohnesorge, D. P. E. Smith, and G. Binnig. 1992. In situ investigations of single living cells infected by viruses. Ultramicroscopy. 42-44:1161-0000; Hörber, J. K. H., W. Häberle, F. Ohnesorge, G. Binnig, H. G. Liebich, C. P. Czerny, H. Mahnel, and A. Mayr. 1992. Investigation of living cells in the nanometer regime with the atomic force microscope. Scanning Microscopy. 6:919-930). Furthermore, the pox viruses used were characterized separately by AFM in an aqueous environment down to the molecular level. Quasi-ordered structural details were resolved on a scale of a few nm where, however, image distortions and artifacts due to multiple tip effects are probably involved--just as in very high resolution (<15-20 nm) images on the cells. Although in a very preliminary manner, initial studies on the mechanical resonance properties of a single living (noninfected) cell, held by the micropipette, have been performed. In particular, frequency response spectra were recorded that indicate elastic properties and enough stiffness of these cells to make the demonstrated rapid scanning of the imaging tip plausible. Measurements of this kind, especially if they can be proven to be cell-type specific, may perhaps have a large potential for biomedical applications. Images of these living cells were also recorded in the widely known (e.g., Radmacher, M., R. W. Tillmann, and H. E. Gaub. 1993. Imaging viscoelasticity by force modulation with the atomic force microscope. Biophys. J. 64:735-742) force modulation mode, yet at one low modulation frequency of approximately 2 kHz. (Note: After the cells were attached to the pipette by suction, they first deformed significantly and then reassumed their original spherical shape, which they also acquire when freely suspended in solution, to a great extent with the exception of the portion adjusting to the pipette edge geometry after approximately 0.5-1 h, which occurred in almost the same manner with uninfected cells, and those that had been infected several hours earlier. This seems to be a process which is at least actively supported by the cellular cytoskeleton, rather than a mere osmotic pressure effect induced by electrolyte transport through the membrane. Furthermore, several hours postinfection (p.i.) infected cells developed many optically visible refraction effects, which appeared as small dark spots in the light microscope, that we believed to be the regions in the cell plasma where viruses are assembled; this is known from the literature on electron microscopy on pox-infected cells and referred to there as "virus factories" (e.g., Moss, B. 1986. Replication of pox viruses. In Fundamental Virology, B. N. Fields and D. M. Knape, editors. Raven Press, New York. 637-655). Therefore, we assume that the cells stay alive during imaging, in our experience for approximately 30-45 h p.i.).     

The distribution of alpha-helix propensity along the polypeptide chain is not conserved in proteins from the same family.

We address the question of whether the distribution of secondary structure propensities of the residues along the polypeptide chain (denominated here as secondary structure profiles) is conserved in proteins throughout evolution, for the particular case of alpha-helices. We have analyzed by CD the conformation of peptides corresponding to the five alpha-helices of two alpha/beta parallel proteins (ComA and Ara). The large alpha-helical population of peptide ComA-4 detected by CD in aqueous solution has been confirmed by NMR. These proteins are members of the CheY and P21-ras families, respectively, which have been studied previously in the same way (Muñoz V, Jiménez MA, Rico M, Serrano L, 1995, J Mol Biol 245:275-296). Comparison of the helical content of equivalent peptides reveals that protein alpha-helix propensity profiles are not conserved. Some equivalent peptides show very different helical populations in solution and this is especially evident in very divergent proteins (ComA and CheY). However, all the peptides analyzed so far adopted an important population of helical conformations in the presence of 30% trifluoroethanol, indicating that there could be a conserved minimal requirement for helical propensity.     

Single-molecule dataset (SMD): a generalized storage format for raw and processed single-molecule data

Background

Single-molecule techniques have emerged as incisive approaches for addressing a wide range of questions arising in contemporary biological research [Trends Biochem Sci 38:30–37, 2013; Nat Rev Genet 14:9–22, 2013; Curr Opin Struct Biol 2014, 28C:112–121; Annu Rev Biophys 43:19–39, 2014]. The analysis and interpretation of raw single-molecule data benefits greatly from the ongoing development of sophisticated statistical analysis tools that enable accurate inference at the low signal-to-noise ratios frequently associated with these measurements. While a number of groups have released analysis toolkits as open source software [J Phys Chem B 114:5386–5403, 2010; Biophys J 79:1915–1927, 2000; Biophys J 91:1941–1951, 2006; Biophys J 79:1928–1944, 2000; Biophys J 86:4015–4029, 2004; Biophys J 97:3196–3205, 2009; PLoS One 7:e30024, 2012; BMC Bioinformatics 288 11(8):S2, 2010; Biophys J 106:1327–1337, 2014; Proc Int Conf Mach Learn 28:361–369, 2013], it remains difficult to compare analysis for experiments performed in different labs due to a lack of standardization.

Results

Here we propose a standardized single-molecule dataset (SMD) file format. SMD is designed to accommodate a wide variety of computer programming languages, single-molecule techniques, and analysis strategies. To facilitate adoption of this format we have made two existing data analysis packages that are used for single-molecule analysis compatible with this format.

Conclusion

Adoption of a common, standard data file format for sharing raw single-molecule data and analysis outcomes is a critical step for the emerging and powerful single-molecule field, which will benefit both sophisticated users and non-specialists by allowing standardized, transparent, and reproducible analysis practices.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0429-4) contains supplementary material, which is available to authorized users.     

On the supramolecular organization of gramicidin channels. The elementary conducting unit is a dimer.

The question, whether the conducting channels formed by the linear gramicidins are dimers (as is generally believed) or tetramers (as has been recently proposed [Stark G., M. Strässle, and Z. Takacz. 1986. J. Membr. Biol. 89:23-37; Strässle, M., G. Stark, M. Wilhelm, P. Daumas, F. Heitz, and R. Lazaro. 1989. Biochim. Biophys. Acta. 980:305-314]) has been addressed in single-channel experiments. The experimental approach was based on the ability of electrophysiological (single-channel) experiments to resolve the number of hybrid channel types that could form between gramicidin A or C and O-pyromellityl-gramicidin A or C (in which a pyromellitic acid residue has been esterified to the ethanolamine-OH group [Apell, H.-J., E. Bamberg, H. Alpes, and P. Läuger. 1977. J. Membr. Biol. 31:171-188]). The presence of the bulky, negatively charged pyromellityl group at the channel entrances endows the hybrid channels with characteristically different features and thus facilitates the resolution of the different hybrid channel types. Only two hybrid channel types were detected, indicating that the conducting channels are membrane-spanning dimers. There was likewise no evidence for lateral association between conducting channels and nonconducting monomers. These results can be reconciled with those of Stark et al. (op. cit.) if gramicidin channel formation involves a (slow) folding into beta 6.3-helical monomers followed by the dimerization step.     

Endonuclease G from mammalian nuclei is identical to the major endonuclease of mitochondria.

Two Mg(2+)-dependent DNA endonucleases have been isolated from mammalian cells which have a strong preference to nick within long tracts of guanine residues in vitro. One endonuclease activity is mitochondrial (mt). The other endonuclease, called Endonuclease G, is associated with isolated nuclei, and is released when the nuclear chromatin is exposed to moderate ionic strength. Our laboratory has previously purified the mt endonuclease to near homogeneity from mitochondria of bovine heart and reported the enzyme to be a homodimer of a approximately 29 kDa polypeptide [Cummings, O. W. et al. (1987) J. Biol. Chem., 262, 2005-2015]. Although the purified mt endonuclease will extensively fragment M13 viral ssDNA and plasmid dsDNAs in vitro, the enzyme displays an unusually strong preference to nick within a (dG)12:(dC)12 sequence tract which resides just upstream from the origin of DNA replication in the mitochondrial genome. The nuclear Endonuclease G first identified from its selective targeting of several (dG)n:(dC)n tracts in vitro (where N = 3-29), was subsequently purified from calf thymus nuclei and shown to be a homodimer of a approximately 26-kDa polypeptide [Côté, J. et al. (1989) J. Biol. Chem., 264, 3301-3310]. In the present study, we find that Endonuclease G partially purified from calf thymus nuclei will extensively degrade both viral ss- and dsDNAs in vitro, and that the enzyme possesses biochemical properties and specificity for nucleotide sequences in DNA that are strongly related or identical to those of the mt endonuclease. These findings and the discovery of sequence identity between the proteins strengthen the conclusion that the nuclear Endonuclease G is the same enzyme as the mt endonuclease.     

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose,bisected hybrid,and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.Abbreviations Con A Concanavalin A with unspecified metal ion content - CMPL Con A with Mn²⁺ and Ca²⁺ at the S1 and S2 sites respectively, in the locked conformation [12]; trisaccharide1, 3,6-di-O-(-d-mannopyranosyl)-d-mannose - -MDM methyl -d-mannopyranoside - NMRD nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, 1/T₁, of solvent protons