A molecular mechanism for stabilization of learning-induced synaptic modifications

Olfaction is a principal sensory modality in rodents, and rats quickly learn to discriminate between odors and to associate odor with reward. Here we show that such olfactory discrimination (OD) learning consists of two phases with distinct cellular mechanisms: an initial NMDAR-sensitive phase in which the animals acquire a successful behavioral strategy (rule learning), followed by an NMDAR-insensitive phase in which the animals learn to distinguish between individual odors (pair learning). Rule learning regulates the composition of synaptic NMDARs in the piriform cortex, resulting in receptors with a higher complement of the NR2a subunit protein relative to NR2b. Rule learning also reduces long-term potentiation (LTP) induced by high-frequency stimulation of the intracortical axons in slices of piriform cortex. As NR2a-containing NMDARs mediate shorter excitatory postsynaptic currents than those containing NR2b, we suggest that learning-induced regulation of NMDAR composition constrains subsequent synaptic plasticity, thereby maintaining the memory encoded by experience.     

Theory of oligonucleotide stabilization. I. The effect of single-strand stacking

Certain theoretical difficulties present in the analysis of thermal transition properties of short complementary double-stranded RNA oligomers can be resolved in part by introducing the stability of the component single-stranded systems explicitly into the model. The stability constant S of the usual theories is redefined so as to contain double-stranded pairing (τt) and single-stranded stacking (ρ) contributions, and we analyze the statistics of two experimental systems—acid oligo(A) and oligo (A_n·U_n) dimers—to exhibit the underlying stability parameters. We present a fitting procedure to extract values of the heats and entropies of the separated components when the required single-strand data is unreliable or not available. The theory leads to length dependent heats and entropies for short single strands in a natural way, and permits a more accurate assessment of the contribution of partially bonded states in thermal transitions than has previously been possible.     

Thermodynamics and mechanism of cutinase stabilization by trehalose

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2 degrees . Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy-enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding.     

A mechanism of Rap1-induced stabilization of endothelial cell--cell junctions

Activation of Rap1 small GTPases stabilizes cell--cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1-Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ~40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell-cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell--cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.     

Hydrogen bonding versus stacking stabilization by modified nucleobases incorporated in PNA·DNA duplexes

The effects of incorporation of the modified nucleobases, 2,6-diaminopurine (D) (substituting for adenine) and 7-chloro-1,8-naphthyridin-2-(1H)-one (bicyclic thymine, bT) (substituting for thymine), that stabilize PNA·DNA duplex formation by increasing hydrogen bonding and/or base pair stacking interactions have been studied by thermal denaturation in terms of thermodynamics. Although the stabilizing effect of the bT base (in contrast to that of D base) is abolished upon addition of dimethyl formamide, thereby indicating that the stabilization is predominantly due to hydrophobic stacking forces, duplex stabilization was found to be enthalpic for both nucleobases. Increased stabilization (although not fully linearly) was observed with increasing numbers of modified bases, and single base sequence discrimination was only slightly compromised, but showed significant dependence on the sequence context.     

Molecular mechanism of AHSP-mediated stabilization of alpha-hemoglobin

Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two alpha and two beta subunits. Free alpha-hemoglobin (alphaHb) is unstable, and its precipitation contributes to the pathophysiology of beta thalassemia. In erythrocytes, the alpha-hemoglobin stabilizing protein (AHSP) binds alphaHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-alphaHb reveals that AHSP specifically recognizes the G and H helices of alphaHb through a hydrophobic interface that largely recapitulates the alpha1-beta1 interface of hemoglobin. The AHSP-alphaHb interactions are extensive but suboptimal, explaining why beta-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound alphaHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-alphaHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free alphaHb.     

Particle geometry and the chromatographic separation of cells: the simplest case of uniform particles

A theoretical study, using numerical methods, of the problem of packing spherical particles has been carried out in order to design an optimal chromatographic packing material for cell separation. It is concluded that a monodisperse packing of hard beads does not yield a packing in which narrow crevices, impenetrable to cells, are excluded. Allowing a certain degree of compressibility alleviates this problem somewhat.     

Remarkable stabilization of antiparallel DNA triplexes by strong stacking effects of consecutively modified nucleobases containing thiocarbonyl groups

The consecutive arrangement of 2′-deoxy-6-thioguanosines (s⁶Gs) and 4-thiothymidines (s⁴Ts) in antiparallel triplex-forming oligonucleotides (TFOs) considerably stabilized the resulting antiparallel triplexes with high base recognition ability by the strong stacking effects of thiocarbonyl groups. This result was remarkable because chemical modifications of the sugar moieties and nucleobases of antiparallel TFOs generally destabilize triplex structures. Moreover, in comparison with unmodified TFOs, it was found that TFOs containing s⁶Gs and s⁴Ts could selectively bind to the complementary DNA duplex but not to mismatched DNA duplexes or single-stranded RNA.     

A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.

Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants.     

Selective stabilization of retinotectal synapses by an activity-dependent mechanism

How does each ingrowing retinal fiber select the right spot in the overall retinotopic projection? Chemospecific surface interactions appear to be sufficient only to organize a crude retinotopic map on the tectum during regeneration of the optic nerve of goldfish. Precise retinotopic ordering is achieved via an activity-dependent stabilization of appropriate synapses, based on the correlated activity of neighboring ganglion cells of the same receptive field type in the retina. Four treatments have been found to block the sharpening process: 1) blocking activity of the ganglion cells with intraocular tetrodotoxin (TTX); 2) rearing in total darkness; 3) correlated activation of all ganglion cells via stroboscopic illumination in a featureless environment; 4) block of retinotectal synaptic transmission with alpha-bungarotoxin. These experiments support a role for normal visually driven activity in sharpening the diffuse projection, and demonstrate that the correlated activity of the optic fibers interacts within the postsynaptic cells, probably through the summation of excitatory postsynaptic potentials. Intraocular TTX experiments suggest that a similar mechanism may drive both the formation of ocular dominance patches in fish tectum and kitten visual cortex and the segregation of different receptive field types in the lateral geniculate nucleus. Thus, it may be a general mechanism whereby the diffuse projections of early development are brought to a mature level of organization.     

Molecular mechanism of polyethylene glycol mediated stabilization of protein

The effect of different molar ratios of polyethylene glycol (PEG) on the conformational stability of protein, bovine serum albumin (BSA), was studied. The binding of PEG with BSA was observed by fluorescence spectroscopy by measuring the fluorescence intensity after displacement of PEG with chromophore ANS and had further been confirmed by measuring the intrinsic fluorescence of tryptophan residues of BSA. Co-lyophilization of BSA with PEG at optimum BSA:PEG molar ratio led to the formation of the stable protein particles. Circular dichroism (CD) spectroscopy study suggested that a conformational change had occurred in the protein after PEG interaction and demonstrated the highest stability of protein at the optimum BSA:PEG molar ratio of 1:0.75. Additional differential scanning calorimetry (DSC) study suggested strong binding of PEG to protein leading to thermal stability at optimum molar ratio. Molecular mechanism operating behind the polyethylene glycol (PEG) mediated stabilization of the protein suggested that strong physical adsorption of PEG on the hydrophobic core of the protein (BSA) along with surface adsorption led to the stability of protein.     

Possible mechanism for the dynamic stabilization of protein structure

There is an apparent contradiction between the long lifetime of the metastable structure of native proteins and the high rate of structural fluctuations, which result from the small activation energy required to change the native conformation.In this paper we point out that the observed stability of proteins is not a consequence of large potential barriers, but a result of the continuous reconstitution of the degraded structure by chain propagation. Polypeptide chains of proteins having naturally selected amino-acid sequences have regenerative ability which ensures the long lifetime of the native structure by making most of the fluctuations reversible.A simple calculation shows that in a certain fluctuation of an average protein molecule the probability of denaturation is less than 10⁻²⁵, therefore even the most rapid, picosecond time scale fluctuations cause spontaneous denaturation only in million year time scale. Hence, the generally observed spontaneous denaturation in vitro is rather a consequence of covalent structure modification or intermolecular interactions than a result of an intramolecular interconversion from the native conformation to another conformation.     

Nebulin regulates actin filament lengths by a stabilization mechanism

Efficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of nebulin (mini-nebulin). When endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either matching or longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous nebulin). Thus, nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.     

Additional stabilization of stem bromelain coupled to a thermosensitive polymer by uniform orientation and using polyclonal antibodies

Stem bromelain was covalently coupled to a thermosensitive polymer of N-isopropylacrylamide (p(NIPAm)) either through the amino groups of the enzyme (randomly coupled) or via the lone oligosaccharide chain (uniformly coupled). The enzyme coupled via the oligosaccharide chain exhibited better access to the substrate casein as compared to the preparation in which the amino groups formed the point of contact between the enzyme and the polymer. Native bromelain exhibited a pH optimum of 8.0 and a broad pH-activity profile. The polymer-coupled preparations exhibited broader pH-activity profiles and shifting of pH optimum to 10.0 at 35°C. At 25°C, the shifting of pH optimum was observed for the randomly coupled enzyme only. The temperature-activity profiles of bromelain coupled to p(NIPAm) also showed appreciable broadening and the preparations retained greater fraction of maximum activity above the temperature optimum. The optimum temperature of the uniformly oriented preparation also rose to 70°C. Inactivation rates of the polymer-coupled bromelain were remarkably low at 60°C as compared to the native protease, and binding of antibromelain antibodies improved the resistance to inactivation of the polymer-coupled preparations. The cleavage patterns of hemoglobin and IgG by the native bromelain and the polymer-coupled preparations were comparable. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 3, pp. 375–382.     

A mathematical analysis of enzyme stabilization by a series-type mechanism: Influence of chemical modifiers

A series-type enzyme deactivation model is utilized to theoretically analyze and to quantify the effect of chemical modifier concentration on the eventual level of enzyme activity stabilization, alpha(2). An increase in the concentration of phosphate ion and NADP increases alpha(2) for the enzymes studied. One example of each enzyme deactivation is given wherein the introduction of chemical modifiers changes the deactivation mechanism from a single-step to a series-type mechanism, and from a series-type to a single-step mechanism. Simple empirical equations are proposed to quantify the effect of chemical modifier concentration on alpha(2).