Thermal unfolding of human high-density apolipoprotein A-1: implications for a lipid-free molten globular state.

Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximately 7, by using differential scanning calorimetry and circular dichroism. At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint Tm 57 +/- 1 degree C, calorimetric enthalpy deltaH(Tm)= 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy deltaHv(Tm) approximately 32.5 kcal/mol, and cooperativity deltaHv(Tm)/deltaH(Tm) approximately 0.16. The enthalpy deltaH(Tm) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximately 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal noncoincident melting of the secondary and tertiary structural elements and indicate a well-defined secondary structure but a largely melted tertiary structure for apoA-1 at approximately 37 degrees C and pH 7. This suggests a molten globular-like state for lipid-free apoA-1 under near-physiological conditions. Our results suggest that in vivo lipid binding by apoA-1 may be mediated via the molten globular apolipoprotein state in plasma.

Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells

Using a new mAb raised against the mouse neuroepithelium, we have identified and cDNA-cloned prominin, an 858-amino acid-containing, 115-kDa glycoprotein. Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from Caenorhabditis elegans predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, our results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related.

Heterothermal acclimation: An experimental paradigm for studying the control of thermal acclimation in crabs

A method for the study of the control of the attainment of thermal acclimation has been applied to the crabs, Cancer pagurus and Carcinus maenas. Crabs were heterothermally acclimated by using an anterior–posterior partition between two compartments, one at 8°C and the other at 22°C. One compartment held a three-quarter section of the crab including the central nervous system (CNS), eye stalks, and ipsilateral legs; the other held a quarter section including the contralateral legs. Criteria used to assess the acclimation responses were comparisons of muscle plasma membrane fatty acid composition and “fluidity.” In both species, the major fatty acids of phosphatidylcholine were 16:0, 18:1, 20:5, and 22:6, whereas phosphatidylethanolamine contained significantly less 16:0 but more 18:0; these fatty acids comprised 80% of the total. Differences in fatty acid composition were demonstrated between fractions obtained from the ipsilateral and contralateral legs from the same heterothermally acclimated individual. In all acclimation states (except 22CNS, phosphatidylcholine fraction), membrane lipid saturation was significantly increased with acclimation at 22° as compared with 8°C. Membrane fluidity was determined by using 1,3-diphenyl-1,3,5 hexatriene (DPH) fluorescence polarization. In both species, membranes from legs held at 8° were more fluid than from legs held at 22°C irrespective of the acclimation temperature of the CNS. Heterothermal acclimation demonstrated that leg muscle membrane composition and fluidity respond primarily to local temperature and were not predominately under central direction. The responses between 8°C- and 22°C-acclimated legs were more pronounced when the CNS was cold-acclimated, so a central influence cannot be excluded.

Efficient construction of long DNA duplexes with internal non-Watson–Crick motifs and modifications

We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson–Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert (∼20 bp) containing the modification or non-Watson–Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields (∼60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid–protein interactions.