Studying the assembly of multicomponent protein and ribonucleoprotein complexes using surface plasmon resonance

The assembly of large macromolecular complexes is an important aspect of cellular organization and metabolism. Interactions involving such complexes in principle follow the same rules as the interactions between single proteins or other macromolecules and can therefore be investigated using similar approaches. We have developed protocols employing standard surface plasmon resonance technology that allow the investigation of interactions involving complex macromolecular structures. The principal experimental challenges arise from the possibility of parallel reactions where partially assembled or dissociated subcomplexes form a significant proportion of the molecule population and from an increased likelihood of unspecific binding events owing to the larger surface and statistically higher number of charged areas on multisubunit assemblies. Ways to experimentally avoid or, where this is not possible, to control for these complications are discussed.     

Adaptation to high temperatures through macromolecular dynamics by neutron scattering

Work on the relationship between hyperthermophile protein dynamics, stability and activity is reviewed. Neutron spectroscopy has been applied to measure and compare the macromolecular dynamics of various hyperthermophilic and mesophilic proteins, under different conditions. First, molecular dynamics have been analyzed for the hyperthermophile malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, the lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The neutron scattering approach has provided independent measurements of the global flexibility and structural resilience of each protein, and it has been demonstrated that macromolecular dynamics represents one of the molecular mechanisms of thermoadaptation. The resilience was found to be higher for the hyperthermophilic protein, thus ensuring similar flexibilities in both enzymes at their optimal activity temperature. Second, the neutron method has been developed to quantify the average macromolecular flexibility and resilience within the natural crowded environment of the cell, and mean macromolecular motions have been measured in vivo in psychrophile, mesophile, thermophile and hyperthermophile bacteria. The macromolecular resilience in bacteria was found to increase with adaptation to high temperatures, whereas flexibility was maintained within narrow limits, independent of physiological temperature for all cells in their active state. Third, macromolecular motions have been measured in free and immobilized dihydrofolate reductase from Escherichia coli. The immobilized mesophilic enzyme has increased stability and decreased activity, so that its properties are changed to resemble those of a thermophilic enzyme. Quasi-elastic neutron scattering measurements have also been performed to probe the protein motions. Compared to the free enzyme, the average height of the activation free energy barrier to local motions was found to be increased by 0.54 kcal.mol(-1) in the immobilized dihydrofolate reductase, a value that is of the same order as expected from the theoretical rate equation.     

Adaptation to extreme environments: macromolecular dynamics in bacteria compared in vivo by neutron scattering

Mean macromolecular dynamics was quantified in vivo by neutron scattering in psychrophile, mesophile, thermophile and hyperthermophile bacteria. Root mean square atomic fluctuation amplitudes determining macromolecular flexibility were found to be similar for each organism at its physiological temperature (~1 Å in the 0.1 ns timescale). Effective force constants determining the mean macromolecular resilience were found to increase with physiological temperature from 0.2 N/m for the psychrophiles, which grow at 4°C, to 0.6 N/m for the hyperthermophiles (85°C), indicating that the increase in stabilization free energy is dominated by enthalpic rather than entropic terms. Larger resilience allows macromolecular stability at high temperatures, while maintaining flexibility within acceptable limits for biological activity.     

Comparative studies on covalent and noncovalent immobilization of protein molecules on the surface of liposomes

Methods of enzyme molecules covalent immobilization on the surface of liposomes are suggested. The methods permit more protein molecules to be bound than traditional methods of noncovalent immobilization by means of adsorption or incorporation. The liposome membranes preserve their integrity during immobilization. At the same tame, the enzyme bound with the liposome surface via the “spacer” groups completely preserves its ability to interact with a specific macromolecular compound.     

Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme

The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism.     

High-level overproduction of His-tagged Tth DNA polymerase in Thermus thermophilus

A new plasmid for the overexpression of His-tagged thermozymes in Thermus thermophilus was developed. With this plasmid, soluble and active histidine-tagged DNA polymerase from T. thermophilus was overproduced in larger amounts in the thermophile than in Escherichia coli. The protein purified from the thermophile was active in PCR.     

High-Level Overproduction of His-Tagged Tth DNA Polymerase in Thermus thermophilus

A new plasmid for the overexpression of His-tagged thermozymes in Thermus thermophilus was developed. With this plasmid, soluble and active histidine-tagged DNA polymerase from T. thermophilus was overproduced in larger amounts in the thermophile than in Escherichia coli. The protein purified from the thermophile was active in PCR.     

Conformational studies on the inactivation of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU

Among the various proposals that have been made in attempting to explain the ability of thermophiles to reproduce at high temperatures, there is no doubt that obligate and extreme thermophiles synthesize proteins (and other molecules) that have sufficient intrinsic molecular stability to withstand increased thermal stress. In contrast, the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU has been shown to be quite thermolabile in vitro. Thermal inactivation is not due to loss of bound NAD+. It has also been shown that the enzymatic activity can be thermostabilized in vitro by increased ionic strength. As previously reported [J. W. Crabb, A. L. Murdock, and R. E. Amelunxen (1975) Biochem. Biophys. Res. Commun. 62, 627; (1977) Biochemistry 16, 4840], the enzyme loses 94-97% of enzymatic activity after heat treatment at 55 degrees C for 5 min in 0.05 M sodium phosphate buffer (pH 7.1); however, by increasing the ionic strength to 1.8, complete protection was conferred at this temperature. Gel-filtration chromatography has been used to study the initial dissociation and subsequent aggregation of the glyceraldehyde-3-phosphate dehydrogenase after thermal inactivation. Aggregation occurs when the enzyme is heated at 50 degrees or 55 degrees C. Loss of enzymatic activity is correlated with changes in the tertiary structure as measured by the near-uv CD spectrum of the enzyme following heat inactivation, with essential disappearance of the peaks at 263 and 296 nm, and a blue shift of the far-uv spectrum, which is a measure of secondary structure. Estimation of secondary structure of the unheated protein from the far-uv CD data showed the enzyme contains approximately 26% alpha-helix, approximately 21% beta-structure, and approximately 53% disordered structure. Heat treatment at various temperatures resulted in only slight changes of the estimated secondary structure. Increased ionic strength prevents thermal alteration of the CD spectrum in both near- and far-uv regions. The data support the previous proposal that thermolabile enzymes such as the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile B. coagulans are thermostabilized in vivo mainly by the intracellular charged macromolecular environment.     

Replacement of the active surface of a thermophile protein by that of a homologous mesophile protein through structure-guided 'protein surface grafting'

Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.     

Effects of a bulking agent on the reaction rate of thermophilic sewage sludge composting

The effects of a bulking agent on the reaction rate of sewage sludge composting was investigated quantitatively. Larger amounts of rice husks added as the bulking agent yielded a higher CO₂ evolution rate and a larger number of thermophiles per unit dry mass of raw sludge. The specific CO₂ evolution rate estimated for each thermophile was almost the same, however, irrespective of the mixing ratio of rice husks. Since the calculation showed that the composting solid particles were aerobic, we suggest that the addition of a bulking agent may increase air-solid interfaces where microorganisms can selectively grow to make colonies.     

Archaeal RecA homologues: different response to DNA-damaging agents in mesophilic and thermophilic Archaea

Two archaeal proteins, RadA and RadB, share similarity with the RecA/Rad51 family of recombinases, with RadA being the functional homologue. We have studied and compared the RadA and RadB proteins of mesophilic and thermophilic Archaea. In growing cells, RadA levels are similar in mesophilic Methanococcus species and the hyperthermophile Methanococcus jannaschii. Treatment of cells with mutagenic agents (methylmethane sulfonate or UV light) increased the expression of RadA (as evidenced by higher levels of both mRNA and protein) in all organisms tested, but the increase was greater in the mesophiles than in the thermophiles M. jannaschii and Sulfolobus solfataricus. Recombinantly expressed RadA proteins from the mesophile M. voltae and the thermophile M. jannaschii were similar in their ATPase- and DNA-binding activities. All the data are consistent with proposals that RadA plays the same role as eukaryotic Rad51. Surprisingly, the data also suggested that the thermophiles do not need more RadA protein or activity than the mesophiles. On the other hand, RadB is not coregulated with RadA, and its role remains unclear. Neither RadA nor RadB from a mesophile or from a thermophile rescued the UV-sensitive phenotype of an Escherichia coli recA- host.     

Chaperonin function--effects of crowding and confinement

Chaperonins assist in the acquisition of native protein structure in the cell by providing a shielded environment for a folding polypeptide chain, generated by the interior surface of their cylindrical structure. The folding chain is isolated from the highly crowded cytoplasm, but at the same time confined within the chaperonin folding cage. Both confinement and macromolecular crowding can affect folding kinetics and yields, the modus operandi of chaperonins and their interaction with their protegés. Recent experimental data, as well as computer simulations, provide increasing evidence that the particular physico-chemical conditions prevailing in the cellular interior have to be taken into account when trying to unravel the processes of cellular protein folding.     

Selective utilization of 2-thioribothymidine- and ribothymidine-containing tRNAs by the protein synthetic systems of Thermus thermophilus HB 8 depending on the environmental temperature

An extreme thermophile, Thermus thermophilus HB 8, contains two types of tRNAs, T- and S2T-containing tRNAs. Their relative content changes depend on the growth temperature of the bacterial cells (1-3). To elucidate the reason why the extreme thermophile possesses the two types of tRNAs, an attempt was made to clarify how these tRNAs are utilized in in vivo protein synthetic systems of the bacteria cultured at different temperatures. First, a method was developed to isolate active polysomes from the thermophile cells cultured at 55 degrees C, 65 degrees C, and 77 degrees C. Then, tRNAs were separated from the polysomes and the T- and S2T-contents of the tRNAs were determined by HPLC. The relative content of S2T-tRNAs in the polysomes from 77 degrees C cells was much higher than that in bulk tRNAs from whole cells cultured at the same temperature, but the situation was reversed in 50 degrees C cells. These results clearly show that the protein synthetic systems of the thermophile have some selection mechanism to utilize either T- or S2T-containing tRNAs preferentially depending on the environmental temperature.     

Temporal variations in growth and reproduction of Tedania anhelans and Chondrosia reniformis in the North Adriatic Sea

Most works concerning growth and reproduction of Mediterranean sponges have been performed in the oligotrophic western Mediterranean while little is known about sponge dynamics in the North-western Adriatic Sea, a basin characterized by low winter temperature and eutrophy. In order to deepen our understanding of sponges in the North Adriatic Sea and verify how its peculiar trophic and physical conditions affect sponge life cycles, temporal trend of sponge cover (%) and reproductive timing of Chondrosia reniformis and Tedania (Tedania) anhelans were studied over a 1-year period looking for a possible relation with variations of temperature or food availability. In C. reniformis, although little variations of sponge cover were evidenced around the year, the number of individuals and their size increase during spring. Asexual reproduction, via drop-like propagules, mainly occurs in spring and summer, while sexual reproduction is characterized by a maximum number of oocytes in August. T. anhelans progressively grows from spring to summer and develops propagules on its surface that reach their maximum size in July. In autumn, the sponge undergoes a process of progressive shrinkage and almost disappears in winter when temperature reaches 7–8°C. Larvae occur during summer. In the North Adriatic Sea sponges have larger sizes, higher density and a wider period of oocytes production compared with the same species from the Mediterranean Sea, suggesting these differences could be due to high food availability characterizing the eutrophic Adriatic basin. On the contrary, the sharp water temperature variations and the very low winter temperature, 5–6°C lower than what has been reported for the Mediterranean Sea, regulate temporal variations in abundance and cause the disappearance of thermophile species during winter.     

Excluded volume as a determinant of macromolecular structure and reactivity

The effect of excluded volume on the thermodynamic activity of globular macromolecules and macromolecular complexes in solution is studied in the hard-particle approximation. Activity coefficients are calculated as a function of the fraction of total volume occupied by macromolecules using relations obtained from scaled particle and lattice models. Significant and readily observable effects are predicted to occur as the fraction of volume occupied by globular macromolecules increases, including the following: (i) Compact quasi-spherical macromolecular conformations become increasingly energetically favored over extended anisometric conformations. (ii) Self- and heteroassociation processes are enhanced, particularly those leading to the formation of compact quasi-spherical aggregates. (iii) Depending upon the details of the reaction mechanism, the rate of an enzyme-catalyzed reaction may monotonically decrease, go through a maximum, or exhibit more complex behavior. A given degree of volume occupancy by larger macromolecules is predicted to have less effect on the structure and self-association of smaller macromolecules than the same degree of volume occupancy by smaller macromolecules has on the structure and self-association of larger macromolecules.     

Poly(vinylpyrrolidone) for bioconjugation and surface ligand immobilization

Poly(vinylpyrrolidone) (PVP), a nonionic and nontoxic polymer with antifouling properties, has been synthesized via RAFT polymerization to obtain thiol-terminated PVP. We demonstrate that when the polymer is adsorbed onto the surface of colloidal silica particles, the terminal thiol groups of PVP remain accessible for chemical modification and lend themselves to the immobilization of ligands. We show that ligand attachment onto the surface via conjugation to PVP is reversible, as the polymer can be desorbed from the surface for conjugate and surface recovery. We present the conjugation of a model peptide and an oligonucleotide to PVP via the polymer terminal thiol and demonstrate that conjugates remain functional in molecular recognition assay. The developed technique offers a novel method to functionalize low-fouling surfaces for a variety of biomedical applications and presents opportunities to use PVP as a macromolecular drug carrier.     

Monomeric malate synthase from a thermophilic Bacillus. Molecular and kinetic characteristics

The molecular weight of malate synthase purified from a thermophilic Bacillus was determined to be 62,000 by sedimentation equilibrium methods, confirming the value obtained earlier by the gel filtration technique. This enzyme and its homologs from other bacteria, which are all monomeric proteins with molecular weights of approximately 60,000. therefore differ from the considerably larger and multimeric malate synthases from yeast, Neurospora crassa, and other eucaryotic microorganisms and plants. Amino acid analysis reveals the thermophile synthase to be relatively rich in glutamic acid and to have a higher content of arginine in comparison with the yeast enzyme. The Bacillus enzyme is an acidic protein with an isoelectric pH of 4.6 and has two sulfhydryl groups titratable with 5,5′-dithiobis(2-nitrobenzoic acid). Its parameters indicative of its overall hydrophobicity and of levels of helicity and turn, which were deduced from the amino acid composition, lie well within the range recorded for a number of mesophile and thermophile enzymes. However, the level of β-sheet structure is considerably lower than that calculated for the yeast synthase; this supports a trend recently observed for certain other thermophile proteins. The synthase isolated from the thermophilic Bacillus appears to be homogeneous by several criteria, although upon electrophoresis in the native state in polyacrylamide it yields two protein bands that are both enzymatically active. Several kinetic characteristics of this enzyme are also reported.     

Ab Initio molecular orbital study on the thermostability of the extreme thermophile tRNA: Role of the base stacking

In extremely thermophilic tRNA, ribosylthymine is replaced by 2-thioribosylthymine at the key site in tRNA. By means of the ab initio molecular orbital (MO) calculation using the 4–31G basis set, we evaluate how this replacement brings about an increment of stacking energy, and how this increment in stacking energy is responsible for the stability of the thermophile tRNA. Calculated stacking energy for G : s²T : Ψ is larger by 4·85 kJ/mol (1·16 kcal/mol) than that for G : T : Ψ. Taking account of the thermodynamical data of yeast tRNAs by Privalov & Filimonov (1978), such an increment in stacking energy seems to considerably contribute to the increase of the midpoint melting temperature (T_m) in the thermophile tRNA, although other factors such as hydrogen bonding, ribose puckering and magnesium ions can not be excluded. It is found that the dispersion force mainly contributes to the stacking energies for G : T : Ψ and G : s²T : Ψ, especially for the latter. From the decomposition of the SCF energy, electrostatic and charge transfer energies are found to contribute to the stabilization of the thermophile tRNA, though the contribution of the former is larger than the latter.     

Reflection coefficients of homopore membranes: effect of molecular size and configuration

Osmotic water flow through membranes with uniform defined pores was measured for a variety of macromolecular solutes. Water flow increased linearly with applied hydrostatic pressure, allowing the effective osmotic pressure of the solutes to be estimated by extrapolation. Reflection coefficients for each solute-membrane combination were calculated and correlated with the ratio of solute size to pore size. For the same mean molecular size, proteins were found to have larger reflection coefficients than dextrans. Molecular rigidity may play a role in this difference in behavior.