Unfolding mechanics of holo- and apocalmodulin studied by the atomic force microscope

The structural stability of calmodulin (CaM) has been investigated previously by chemical and thermal methods. The calcium-loaded form of CaM has been found to be exceptionally stable, because it can be exposed to temperatures >90 degrees C or to a 9 M urea solution without a marked change in its tertiary structure, and is therefore not experimentally accessible for unfolding studies using conventional analytical methods. In this study, we have developed a system for measuring the force for mechanically unfolding CaM using an atomic force microscope (AFM) by stretching the protein from its N- and C-terminal residues; we have been successful in obtaining force versus extension (F-E) curves for both apo and holo forms of CaM. In our experiment, distinguishable F-E curves have been obtained upon stretching of apoCaM and holoCaM to their full extensions. A very low force observed upon stretching of apoCaM indicated a relatively high flexibility of the apo form. On the contrary, a relatively high unfolding force and the appearance of a characteristic force peak were noted during full stretching of holoCaM. The F-E curve of the latter form of CaM most likely reflects a more rigid and probably more organized conformation of holoCaM than that of apoCaM. These experiments confirmed that the AFM is able to clearly distinguish two functionally distinct forms of CaM in terms of their mechanical properties.     

Heat-induced changes in the conformation of alpha- and beta-crystallins: unique thermal stability of alpha-crystallin

Of the crystallin proteins of the lens, the principal subunit of the beta-crystallin, beta B2 (beta Bp), has been considered to be the only heat-stable protein because it does not precipitate upon heating. In our recent investigations, however, we have found that the alpha-crystallin from bovine lenses is not only heat stable but also does not denature at temperatures up to 100 degrees C. Using circular dichroism and fluorescence to monitor the conformational changes of alpha- and beta B2-crystallins upon heating, we found that alpha-crystallin maintains a high degree of structure, whereas the beta B2-crystallin shows a reversible sigmoidal order-disorder transition at about 58 degrees C.     

Structural basis for thermostability in aporubredoxins from Pyrococcus furiosus and Clostridium pasteurianum

The structures of apo- and holorubredoxins from Pyrococcus furiosus (PfRd) and Clostridium pasteurianum (CpRd) have been investigated and compared using residual dipolar couplings to probe the origin of thermostability. In the native, metal (Fe or Zn) containing form, both proteins can maintain native structure at very high temperatures (>70 degrees C) for extended periods of time. Significant changes in either structure or backbone dynamics between 25 and 70 degrees C are not apparent for either protein. A kinetic difference with respect to metal loss is observed as in previous studies, but the extreme stability of both proteins in the presence of metal makes thermodynamic differences difficult to monitor. In the absence of metal, however, a largely reversible thermal denaturation can be monitored, and a comparison of the two apoproteins can offer insights into the origin of stability. Below denaturation temperatures apo-PfRd is found to have a structure nearly identical to that of the native holo form, except immediately adjacent to the metal binding site. In contrast, apo-CpRd is found to have a structure distinctly different from that of its holo form at low temperatures. This structure is rapidly lost upon heating, unfolding at approximately 40 degrees C. A PfRd mutant with the hydrophobic core mutated to match that of CpRd shows no change in thermostability in the metal-free state. A metal-free chimera with residues 1-15 of CpRd and the remaining 38 residues of PfRd is severely destabilized and is unfolded at 25 degrees C. Hence, the hydrophobic core does not seem to be the key determinant of thermostability; instead, data point to the hydrogen bond network centered on the first 15 residues or the interaction of these 15 residues with other parts of the protein as a possible contributor to the thermostability.     

Chromatin and nucleosome structure.

Chromatin nucleosomes (mononucleosomes through pentanucleosomes) have been isolated by staphylococcal nuclease digestion of calf thymus nuclei. The peak value ellipticity is the same for all oligomers, 1900 deg cm2, mol-1 at 280-nm, 23 degrees C. The dh280/dT vs T show a progressive increase in Tm of the main thermal band (73.5 degrees C, monomer; 79 degrees C, pentamer). Very small amounts of free DNA can be observed in the melting profiles, and shoulders at 60 degrees C and 93 degrees C appear and increase in magnitude as the particle size increases. The magnitude of the change, delta[theta]280, increases with oligomer size. This pattern could result from an initial unfolding of an asymmetric assembly of nucleosomes (polynucleosome superhelix) in addition to the denaturation of the internal nucleosome structure, and a subsequent or simultaneous denaturation of the double strand DNA. The extent of this unfolding appears to depend upon the size of the oligomer and therefore implies interactions between asymmetrically assembled neighboring nucleosomes.     

Synthesis of N-[6-(ethylenedioxy)hexyl]biotinamide: a biotinyl aldehyde precursor for labeling hydrazine-modified biomolecules.

Synthetic approaches for obtaining biotinyl aldehydes are described. While such aldehydes have limited shelflife, the acetal derivative, N-[6-(ethylenedioxy)hexyl]biotinamide, IX, was found to be indefinitely stable upon storage at -20 degrees C. Mild acid hydrolysis conveniently unmasks the aldehyde, which can then be used to label hydrazine-tagged biomolecules.     

Spruce budworm antifreeze protein: changes in structure and dynamics at low temperature

Antifreeze proteins (AFPs) prevent the growth of ice, and are used by some organisms that live in sub-zero environments for protection against freezing. All AFPs are thought to function by an adsorption inhibition process. In order to elucidate the ice-binding mechanism, the structures of several AFPs have been determined, and have been shown to consist of different folds. Recently, the first structures of the highly active insect AFPs have been characterized. These proteins have a beta-helix structure, which adds yet another fold to the AFP family. The 90-residue spruce budworm (Choristoneura fumiferana) AFP consists of a beta-helix with 15 residues per coil. The structure contains two ranks of aligned threonine residues (known as the TXT motif), which were shown by mutagenesis experiments to be located in the ice-binding face. In our previous NMR study of this AFP at 30 degrees C, we found that the TXT face was not optimally defined because of the broadening of NMR resonances potentially due to weak oligomerization. We present here a structure of spruce budworm AFP determined at 5 degrees C, where this broadening is reduced. In addition, the 1H-15N NMR dynamics of the protein were examined at 30 degrees C and 5 degrees C. The results show that the spruce budworm AFP is more structured at 5 degrees C, and support the general observation that AFPs become more rigid as the temperature is lowered.     

Scanning electron microscopic investigation of thermal damage of the teeth.

Sound teeth were heated to 200 degrees C and 1300 degrees C and the gradually developing morphological changes have been studied. The cementum structure was destroyed at about 500 degrees C, the enamel structure between 700 and 900 degrees C, whereas dentine preserved its canalicular structure even after the inorganic salts had melted at 900 degrees to 1000 degrees C. At 1300 degrees C the mineral substances of the tooth were melting into atypical, globular formations. Scanning electron microscopic examination of dental residues damaged by high temperature seems valuable from forensic, criminological as well as anthropological aspects, since the origin of the finding can be determined, from a small fragment, the material can be identified with a tooth and conclusions can be drawn concerning the temperature inducing the damage.     

High-flow-rate hydroxylapatites

We have established a novel and simple method for preparing rigidly crystallized or granulated hydroxylapatites, which showed flow rates one order of magnitude higher in column chromatography than those of the Tiselius specimen (1). The essential difference in the procedures from the Tiselius method is that calcium phosphate, the precursor of hydroxylapatite, is generated not at room temperature but at either 45 or 95 degrees C. The capacities of the two hydroxylapatites for binding and fractionating protein, RNA, and DNA upon column chromatography were similar to those of Tiselius's hydroxylapatite. The 95 degrees C specimen can be dried with no change in the properties. Because of the reproducibly observed properties, including the extremely high flow rate with ease in handling as well as low cost and simple procedures with no additives and with no special equipment in preparation, our hydroxylapatites seem to be best so far for routine laboratory use.     

Modern acrylics for post-embedding immunostaining techniques

We describe two methods for rapid processing of biological tissues into LR White acrylic plastic. Both methods make use of LR White's compatibility with small amounts of water, enabling non-osmicated tissue to be only partially dehydrated before infiltration with the plastic, a procedure that improves the sensitivity of post-embedding immunocytochemistry. In addition, both methods are designed to reduce the time for which tissue is exposed to the damaging influence of the plastic monomer, which can cause extraction and sudden shrinkage. The tissue example used in the first method is immersion-fixed, surgically removed human pituitary which, by virtue of its thorough fixation, can be processed quickly at 50 degrees C using catalytic polymerization at room temperature. The concentration of the catalyst is critically set to prevent the temperature rising above 60 degrees C in the tissue blocks. Penetration of immunoperoxidase reagents into 330-nm LR White sections is demonstrated and possible modes of action are discussed. When "lightly" fixed tissue is processed as above, serious polymerization artifacts can result from autocatalysis. A second method, based on the first but employing slower polymerization at 0 degrees C, has therefore been developed. The high level of fine structure that can be retained using this method is illustrated by the demonstration of the trans-tubular Golgi in perfusion-fixed kidney of rat. Biotinylated lectin is localized to cells of the kidney proximal tubule with streptavidin-colloidal gold, to illustrate tissue reactivity. In a second example, the structure of the bacterial cell envelope is shown to be similar in appearance after partial dehydration and LR White embedding to that seen after progressive lowering of temperature, dehydration, and Lowicryl embedding.     

Effects of frozen storage on the structure of sarcocysts in pig muscle and implications in taxonomic studies

The morphology of the cyst wall of Sarcocystis has unique characteristics that can be used in species identification. To find a suitable way to preserve Sarcocystis cyst samples for species identification, by light microscopy and electron microscopy, we recorded the morphological changes in the cysts of Sarcocystis suihominis and Sarcocystis miescheriana from pig muscle, induced by storage at -20 degrees C. Comparisons were made between fresh cysts and those subjected to frozen storage for periods of 3 days, 20 days and 30 days. Results: cyst wall of the two Sarcocystis species appeared unaffected by storage. There was no obvious change in the length, nor in the width of the protrusions after storage (P>0.05), but the structure of the bradyzoite in the sarcocyst was in many cases disintegrated at -20 degrees C in 20 days for S. miescheriana and 30 days for S. suihominis. To our knowledge this is the first report that Sarcocystis cyst in muscle can be stored at -20 degrees C before and remain suitable for ultrastructural morphological study. Consequently, this paper proposes freezing as a convenient storage method for samples used in taxonomic studies of Sarcocystis.     

Effect of temperature on the structure of trout troponin C

Adaptation for life at different temperatures can cause changes in many aspects of an organism. One example is the expression of different protein isoforms in species adapted to different temperatures. The calcium regulatory protein cardiac troponin C (cTnC), from rainbow trout (Oncorhynchus mykiss), is a good model for studying temperature effects, both because of its low physiological temperature and because mammalian cTnC, extensively studied at higher temperatures, can be used for comparison. We determined the structure and studied the backbone dynamics of the regulatory domain of trout cardiac troponin C (ScNTnC) with one Ca(2+) bound at 7 and 30 degrees C, using nuclear magnetic resonance spectroscopy (NMR). The overall fold of the regulatory domain of trout cTnC at both temperatures is similar to the regulatory domain of mammalian (human, bovine, and porcine isoform) cTnC bound to one Ca(2+). By comparing the trout structures at the two temperatures, we identify differences between the positions of the helices flanking the calcium binding loops, and the overall structure at 7 degrees C is more compact than that at 30 degrees C. The structure at 7 degrees C is more similar to the mammalian cTnC, which was determined at 30 degrees C, indicating that they have the same conformation at their respective physiological temperatures. The dynamic properties of the regulatory domain of trout cTnC are similar at the two temperatures that were used in these studies.     

Dynamic properties of bacteriorhodopsin in purple membranes upon heat treatment

Reversible temperature-dependent conformational changes in bacteriorhodopsin of the purple membranes from Halobacterium halobium have been studied by the method of deuterium exchange. A noticeable increase in the mobility of structured peptide groups in bacteriorhodopsin was revealed upon reorganization of the supermolecular structure at about 60 degrees C. In the supermolecular structure formed, bacteriorhodopsin molecules have no contacts with external medium at 75-80 degrees C. Membrane destruction results in a drastic increase in molecular mobility within the narrow temperature range 100-110 degrees C. The effects observed are induced by predenaturation changes in the bacteriorhodopsin structure and rearrangements in the structure of a protein-lipid complex. The temperature dependence of the number of peptide groups involved in reversible conformational rearrangements is in good agreement with the microcalorimetry data.     

Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase

Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30 degrees C). A plot of temperature-induced changes in (1)H-(2)H exchange, the decrease in the absorbance of the alpha-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45 degrees C and the major one around 59 degrees C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52 degrees C, respectively). The transition, which occurs at 39-45 degrees C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52-59 degrees C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7 degrees C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS.     

UV- and CD-spectra of restriction endonuclease EcoRII and DNA-methylase EcoRII

UV- and CD-spectra of homogeneous enzymes have been measured. Extinction coefficients estimated from the UV-spectra are 0.97 for restriction endonuclease EcoRII at 279.5 nm and 1.17 for DNA-methylase EcoRII at 279 nm. As it follows from the CD spectra, both enzymes have a well developed tertiary structure and a highly ordered secondary structure, which consists of 22% alpha-helices, 64% beta-structure and 9% bends for REcoRII and of 44% alpha-helices, 48% beta-structure and 4% bends for MEcoRII. Restriction endonuclease denatures at 50 degrees C, while DNA-methylase denatures at 45 degrees C, with partial reversibility upon cooling.     

The perturbations of the native state of goat alpha-lactalbumin induced by 1,1'-bis(4-anilino-5-naphthalenesulfonate) are Ca2+-dependent.

In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition. Therefore, it can be expected that in both conditions the protein is equally susceptible to interaction with bis-ANS. Nevertheless, we have observed a number of interesting differences in the interaction of the dye with the apo and Ca2+ form. Native apo-GLA binds two bis-ANS molecules and in the complex with bis-ANS, the far-UV circular dichroism (CD) spectrum of apo-GLA becomes similar to that of the protein in the molten globule state. In contrast, native Ca2+-GLA binds five bis-ANS molecules and the far-UV CD spectrum of native Ca2+-GLA is conserved for the complex. In both cases, the high activation energies observed in kinetic experiments indicate that upon binding, large parts of the protein structure have to be reorganized. The reduced perturbation of the protein structure in the presence of Ca2+ can be attributed to local stabilization effects.     

Temperature dependence of the surface topography in dimyristoylphosphatidylcholine/distearoylphosphatidylcholine multibilayers

Simple lipid binary systems are intensively used to understand the formation of domains in biological membranes. The size of individual domains present in the gel/fluid coexistence region of single supported bilayers, determined by atomic force microscopy (AFM), generally exceeds by two to three orders of magnitude that estimated from multibilayers membranes by indirect spectroscopic methods. In this article, the topography of equimolar dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) multibilayers, made of two superimposed bilayers supported on mica surface, was studied by AFM in a temperature range from room temperature to 45 degrees C. In the gel/fluid phase coexistence region the size of domains, between approximately 100 nm and a few micrometers, was of the same order for the first bilayer facing the mica and the top bilayer facing the buffer. The gel to fluid phase separation temperature of the first bilayer, however, could be increased by up to 8 degrees C, most likely as a function of the buffer layer thickness that separated it from the mica. Topography of the top bilayer revealed the presence of lipids in ripple phase up to 38-40 degrees C. Above this temperature, a pattern characteristic of the coexistence of fluid and gel domains was observed. These data show that difference in the size of lipid domains given by AFM and spectroscopy can hardly be attributed to the use of multibilayers models in spectroscopy experiments. They also provide a direct evidence for metastable ripple phase transformation into a gel/fluid phase separated structure upon heating.     

Mutant enrichment of Schizosaccharomyces pombe by inositol-less death.

Enrichment procedures, such as those utilizing inositol-less death, have proven to be extremely powerful for increasing the efficiency of identification of spontaneous mutants in a variety of procaryotic and eucaryotic organisms. We characterized inositol-less death in several widely used strains of the inositol-requiring yeast Schizosaccharomyces pombe and determined conditions under which this phenomenon can be used to enrich for mutants. Conflicting reports in the literature on the effects of inositol starvation upon viability of S. pombe had cast doubt on the suitability of using inositol-less death in a mutant enrichment procedure for this organism. We determined that inositol-less death was strain dependent, with differences in viability of up to 5 orders of magnitude observed between the most-sensitive strain, 972, and the least-sensitive strain, SP837. Inositol-less death was also dependent upon the cell concentration at the time of initiation of starvation. While inositol-less death occurred at all four temperatures tested, the kinetics of death was slower at 16 degrees C than at 23, 30, or 37 degrees C. Inositol-less death was observed during growth in fermentable and nonfermentable carbon sources, although loss of viability in glycerol-ethanol was significantly slower than that in glucose, sucrose, or raffinose. The feasibility of exploiting inositol-less death to enrich for spontaneous mutants was demonstrated by the identification of amino acid auxotrophs, nucleotide auxotrophs, carbon source utilization mutants, and temperature-sensitive mutants. By varying starvation conditions, some mutants were recovered at frequencies as high as 5.7 x 10(-2), orders of magnitude higher than the spontaneous mutation rate.     

Structure-based incorporation of 6-methyl-8-(2-deoxy-beta-ribofuranosyl)isoxanthopteridine into the human telomeric repeat DNA as a probe for UP1 binding and destabilization of G-tetrad structures

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(TTAGGG)n. Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG)2 is approximately 5 nm at 100 mm NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the Tm of the hTR sequence d(TAGGGT)4 from 67.0 degrees C to 36.1 degrees C at physiological conditions (150 mm KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.