Correlations between internal mobility and stability of globular proteins.

The recent work is surveyed which leads to the suggestions that the conformation of globular proteins in solution corresponds to a dynamic ensemble of rapidly interconverting spatial structures, that clusters of hydrophobic amino acid side chains have an important role in the architecture of protein molecules, and that mechanistic aspects of protein denaturation can be correlated with internal mobility seen in the native conformation. These conclusions resulted originally from high resolution 1H nuclear magnetic resonance (NMR) studies of aromatic ring mobility, exchange of interior amide protons and thermal denaturation of the basic pancreatic trypsin inhibitor and a group of related proteins. Various new approaches to further characterize proteins in solution have now been taken and preliminary data are presented. These include computer graphics to outline hydrophobic clusters in globular protein structures, high resolution 1H-NMR experiments at variable hydrostatic pressure and 13C-NMR relaxation measurements. At the present early stage of these new investigations it appears that the hydrophobic cluster model for globular proteins is compatible with the data obtained.     

A tradeoff between protein stability and conformational mobility in homotrimeric dUTPases

Oligomerization directs active site formation in homotrimeric 2'-deoxyuridine triphosphate pyrophosphatases (dUTPases). Stability of the homotrimer is a central determinant in enzyme function. The present comparative studies of bacterial and fruitfly dUTPases with homologous 3D structures by differential scanning microcalorimetry; fluorescence, circular dichorism and infrared spectroscopies, demonstrate that unfolding is a two-state highly cooperative transition in both dUTPases excluding a significantly populated intermediate state of dissociated and folded monomers. The eukaryotic protein is much less resistant against either thermal or guanidine hydrochloride-induced denaturation. Results suggest that hydrophobic packing of the inner threefold channel of the dUTPase homotrimer greatly contributes to stability.     

Thermodynamic characterization of nucleoplasmin unfolding: interplay between function and stability

The unfolding equilibrium of recombinant (rNP) and natural variants of nucleoplasmin (NP) from Xenopus laevis has been analyzed using biochemical and spectroscopic techniques. In the presence of denaturing concentrations of guanidinium salts (GuHCl and GuSCN), both domains, core and tail, of the rNP pentamer unfold as proven using single-carrying tryptophan mutants, whereas urea is remarkably unable to fully unfold rNP. Chemical unfolding is reversible and can be described well as a two-state transition in which the folded pentamer is directly converted to unfolded monomers, with no evidence of (partially) folded monomers. Therefore, rNP dissociates and fully unfolds simultaneously (N 5 <--> 5U). Activation of the protein by hyperphosphorylation is accompanied by a destabilization of the protein oligomer. A comparison of natural NP forms isolated from eggs and oocytes of X. laevis and recombinant NP reveals that natural variants can be fully unfolded by urea and exhibit D 50 (denaturant concentration at the transition midpoint) values lower than that of the nonphosphorylated protein. Progressive phosphorylation of NP correlates with a gradual loss of stability of 6 kcal/mol (oNP) and 10 kcal/mol (eNP), as compared with the nonphosphorylated protein pentamer. These results suggest that the remarkable stability of the recombinant protein is required to cope with the destabilization brought about by its phosphorylation-induced activation.     

Chemical glycosylation: new insights on the interrelation between protein structural mobility, thermodynamic stability, and catalysis

Chemical protein glycosylation was employed to sequentially modulate the structural dynamics of the serine protease alpha-chymotrypsin as evidenced from amide H/D exchange kinetics. The reduction in alpha-CT's structural dynamics at increasing glycan molar contents statistically correlated with the increased thermodynamic stability (T(m)) and reduced rate of enzyme catalysis (k(cat)) exhibited by the enzyme upon chemical glycosylation. Temperature-dependent experiments revealed that native-like structural dynamics and function could be restored for the glycosylated conjugates at temperature values close to their thermodynamic stability suggesting that the concept of "corresponding states" can be extended to glycoproteins. These results demonstrate the value of chemical glycosylation as a tool for studying the role of protein structural dynamics on protein biophysical properties; e.g. enzyme stability and function.     

The left end of IS2: a compromise between transpositional activity and an essential promoter function that regulates the transposition pathway

Cut-and-paste (simple insertion) and replicative transposition pathways are the two classical paradigms by which transposable elements are mobilized. A novel variation of cut and paste, a two-step transposition cycle, has recently been proposed for insertion sequences of the IS3 family. In IS2 this variation involves the formation of a circular, putative transposition intermediate (the minicircle) in the first step. Two aspects of the minicircle may involve its proposed role in the second step (integration into the target). The first is the presence of a highly reactive junction formed by the two abutted ends of the element. The second is the assembly at the minicircle junction of a strong hybrid promoter which generates higher levels of transposase. In this report we show that IS2 possesses a highly reactive minicircle junction at which a strong promoter is assembled and that the promoter is needed for the efficient completion of the pathway. We show that the sequence diversions which characterize the imperfect inverted repeats or ends of this element have evolved specifically to permit the formation and optimal function of this promoter. While these sequence diversions eliminate catalytic activity of the left end (IRL) in the linear element, sufficient sequence information essential for catalysis is retained by the IRL in the context of the minicircle junction. These data confirm that the minicircle is an essential intermediate in the two-step transposition pathway of IS2.     

Relationship between genetic variation in thermal stability and electrophoretic mobility of mouse β-galactosidase

We have examined the relationships between the genetic determinants for mouse beta-galactosidase heat stability and electrophoretic mobility, in order to clarify previous reports indicating that a variation for enzyme heat stability is restricted to kidney while that for electrophoretic mobility is expressed in all tissues. We find that the two phenotypes show concordant strain distributions and cosegregate in genetic crosses. In contrast to a previous report, the thermal stability variation is expressed in all tissues, although the absolute rate of enzyme inactivation is tissue specific. The evidence supports the notion that a single beta-galactosidase structural locus is expressed in all tissues and that the differences in enzyme stability between tissues results from posttranslational enzyme modification.     

Spatial and energy compromise between host and parasite: the Biomphalaria glabrata-Schistosoma mansoni system.

The development of a sporocyst infrapopulation of Schistosoma mansoni within the Biomphalaria glabrata snail is, from a spatial point of view, detrimental to the host's digestive-genital gland complex growth. For mono- and plurimiracidial infections, the digestive gland volumes are, respectively 51 and 24% of those of control snails. Identical reduction of the infected genital gland volume (43% of controls) occurs in both cases. After the prepatent period, the ratio of parasite/digestive gland volumes (P/PDG) remains fairly constant at around 0.60 independent of the miracidial dose infection, indicative of a balanced host-parasite development which is discussed in relation to the spatial and energy constraints of this system.     

Thermal stability of lysozyme as a function of ion concentration: A reappraisal of the relationship between the Hofmeister series and protein stability

Anion and cation effects on the structural stability of lysozyme were investigated using differential scanning calorimetry. At low concentrations (<5 mM) anions and cations alter the stability of lysozyme but they do not follow the Hofmeister (or inverse Hofmeister) series. At higher concentrations protein stabilization follows the well‐established Hofmeister series. Our hypothesis is that there are three mechanisms at work. At low concentrations the anions interact with charged side chains where the presence of the ion can alter the structural stability of the protein. At higher concentrations the low charge density anions perchlorate and iodide interact weakly with the protein. Their presence however reduces the Gibbs free energy required to hydrate the core of the protein that is exposed during unfolding therefore destabilizing the structure. At higher concentrations the high charge density anions phosphate and sulfate compete for water with the protein as it unfolds increasing the Gibbs free energy required to hydrate the newly exposed core of the protein therefore stabilizing the structure.     

Acetylcholinesterase mobility and stability at the neuromuscular junction of living mice

Acetylcholinesterase (AChE) is an enzyme that terminates acetylcholine neurotransmitter function at the synaptic cleft of cholinergic synapses. However, the mechanism by which AChE number and density are maintained at the synaptic cleft is poorly understood. In this work, we used fluorescence recovery after photobleaching, photo-unbinding, and quantitative fluorescence imaging to investigate the surface mobility and stability of AChE at the adult innervated neuromuscular junction of living mice. In wild-type synapses, we found that nonsynaptic (perisynaptic and extrasynaptic) AChEs are mobile and gradually recruited into synaptic sites and that most of the trapped AChEs come from the perijunctional pool. Selective labeling of a subset of synaptic AChEs within the synapse by using sequential unbinding and relabeling with different colors of streptavidin followed by time-lapse imaging showed that synaptic AChEs are nearly immobile. At neuromuscular junctions of mice deficient in alpha-dystrobrevin, a component of the dystrophin glycoprotein complex, we found that the density and distribution of synaptic AChEs are profoundly altered and that the loss rate of AChE significantly increased. These results demonstrate that nonsynaptic AChEs are mobile, whereas synaptic AChEs are more stable, and that alpha-dystrobrevin is important for controlling the density and stability of AChEs at neuromuscular synapses.     

Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function

The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.     

Visualizing the analogy between competitive adsorption and colloid stability to restore lung surfactant function

We investigated a model of acute respiratory distress syndrome in which the serum protein albumin adsorbs to an air-liquid interface and prevents the thermodynamically preferable adsorption of the clinical lung surfactant Survanta by inducing steric and electrostatic energy barriers analogous to those that prevent colloidal aggregation. Chitosan and polyethylene glycol (PEG), two polymers that traditionally have been used to aggregate colloids, both allow Survanta to quantitatively displace albumin from the interface, but through two distinct mechanisms. Direct visualization with confocal microscopy shows that the polycation chitosan coadsorbs to interfacial layers of both Survanta and albumin, and also colocalizes with the anionic domains of Survanta at the air-liquid interface, consistent with it eliminating the electrostatic repulsion by neutralizing the surface charges on albumin and Survanta. In contrast, the PEG distribution does not change during the displacement of albumin by Survanta, consistent with PEG inducing a depletion attraction sufficient to overcome the repulsive energy barrier toward adsorption.     

Organization of work in the honeybee: a compromise between division of labour and behavioural flexibility

Although the caste concept has been central to our understanding of the organization of work in social insect colonies, the concept has been the subject of considerable recent criticism. Theoretically, it has been suggested that temporal castes are too inflexible to allow a colony to rapidly reallocate labour in response to changing conditions. In addition, several authors have suggested that task switching is so prevalent that it precludes even the possibility of a rigidly controlled temporal caste system. This study addresses these two criticisms by presenting and testing a revision of the temporal caste concept that recognizes two categories of tasks: those that require a physiological specialization for their efficient performance, and those that all workers are equally able to perform. Only those tasks requiring a physiological specialization are relevant to the temporal caste concept. Two castes of honeybees were shown to vary in response to increased nectar influx, which requires a physiological specialization, but not to heat stress, which requires no specialization. This work suggests that the organization of work in social insect colonies reflects a compromise between selection for the benefits of division of labour and opposing selection for flexibility in task allocation.     

MicroRNA: the perfect host

To truly understand the biochemistry of cellular systems, the concentrations of the endogenous protein players must be known, and these can be obtained by careful quantification of fluorescent fusion proteins.     

Asymmetries in body part size, mobility, and usage. Relationship between structure and function

Asymmetries in structure (size) and function (usage or mobility) for the upper and lower face, hands, and feet were investigated in 42 left- and right-handed male and female college students. Size measurements were taken from photographs, mobility was rated from videotapes, and usage was assessed by questionnaire. Size asymmetries were typical, and independent of handedness, but were not consistent across body parts; usage asymmetries varied as a function of handedness. No systematic relationships were found between structure and function.     

Histone tails and the H3 alphaN helix regulate nucleosome mobility and stability

Nucleosomes fulfill the apparently conflicting roles of compacting DNA within eukaryotic genomes while permitting access to regulatory factors. Central to this is their ability to stably associate with DNA while retaining the ability to undergo rearrangements that increase access to the underlying DNA. Here, we have studied different aspects of nucleosome dynamics including nucleosome sliding, histone dimer exchange, and DNA wrapping within nucleosomes. We find that alterations to histone proteins, especially the histone tails and vicinity of the histone H3 alphaN helix, can affect these processes differently, suggesting that they are mechanistically distinct. This raises the possibility that modifications to histone proteins may provide a means of fine-tuning specific aspects of the dynamic properties of nucleosomes to the context in which they are located.     

Swiss-Prot: juggling between evolution and stability

We describe some of the aspects of Swiss-Prot that make it unique, explain what are the developments we believe to be necessary for the database to continue to play its role as a focal point of protein knowledge, and provide advice pertinent to the development of high-quality knowledge resources on one aspect or the other of the life sciences.     

Continuous assessment of knee function and patient mobility

A technique is described for measuring and analysing the load profile of the knees during daily routine. The method has been used on patients with chronic arthritis of the knee, both before and after joint replacement. Results are presented, and the logistics of carrying out such work are discussed. The method is applicable to joints other than the knee, diseases other than arthritis, and treatments other than joint replacement.