The conformational properties of the polyproline I (PPI) helix of oligoprolines toward heating were examined. Oligoproline H-Pro(12)-NH(2) served as a model which adopts in n-PrOH a pronounced PPI conformation with all cis amide bonds, whereas a polyproline II (PPII) conformation with all trans amide bonds is predominant in pure aqueous buffer. CD spectroscopic studies revealed that a conformational change from the PPI to the PPII helix takes place upon heating and back to the PPI helix upon cooling. This conformational transition cycle is characterized by a strong hysteresis. With a quantitative fitting of the experimentally observed hysteresis loops by a newly developed iterative integration with different starting conditions, kinetic and thermodynamic parameters for the transition from the PPI to the PPII helical conformation were determined. The transition is as expected for cis-trans isomerizations of amide bonds comparatively slow (k = 0.003 s(-1) at 80 °C) and characterized by an activation energy E(a) of 81.1 ± 3.6 kJ mol(-1). Thermodynamically, the transition from the PPI to the PPII helix is characterized by a positive standard enthalpy (ΔH(0) = 33.5 ± 2.1 kJ min(-1)) and a positive standard entropy (ΔS(0) = 102 ± 6.6 J mol(-1) K(-1)).
A procedure was developed for purifying the globular domain NC1 of basement membrane collagen from collagenase digests of a variety of tissues. The globule (Mr = 170,000) is a hexameric structure originating from two collagen IV molecules that are cross-linked at their COOH-terminal ends. Dissociation into subunits derived from alpha 1(IV) and alpha 2(IV) chains occurs at a pH below 4 and after denaturation (8 M urea). The subunits obtained include monomers (Mr = 28,000) and two different dimers (Da,Db) which are connected by disulfide bonds (Db) and/or nonreducible bonds (Da). Almost perfect reconstitution to hexamers is obtained in neutral buffer with mixtures of the subunits or purified dimers but not with purified monomers. Stabilization by dimer formation and other physical data suggest conformationally distinct segments within the subunits, which is also supported by a repeating subdomain structure deduced from cDNA sequences. Monocline crystals of NC1 give a sufficiently detailed X-ray diffraction pattern that should permit elucidation of the three-dimensional structure of the hexamer. Antibodies raised against the globular domain react with all subunits and mainly recognize epitopes stabilized by internal disulfide bridges and/or the hexameric assembly. Immunoprecipitation tests with these antibodies demonstrated a slightly larger subunit size of NC1 in PYS-2 cell culture and the rapid release of precursor-specific segments prior to secretion from the cells. Autoantibodies against mouse tumor NC1 were produced in mice and were detected both in the blood and as tissue-bound forms (kidney, lung). The autoantibody response is accompanied by certain pathological alterations mimicking Goodpasture's syndrome. The possible relationship between the two diseases is substantiated by reaction of Goodpasture antisera with the globular domain obtained from various tissue sources.
