The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell–cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.

Measured by ultra-slow scanning calorimetry and isothermal circular dichroism, human lung collagen monomers denature at 37°C within a couple of days. Their unfolding rate decreases exponentially at lower temperature, but complete unfolding is observed even below 36°C. Refolding of full-length, native collagen triple helices does occur, but only below 30°C. Thus, contrary to the widely held belief, the energetically preferred conformation of the main protein of bone and skin in physiological solution is a random coil rather than a triple helix. These observations suggest that once secreted from cells collagen helices would begin to unfold. We argue that initial microunfolding of their least stable domains would trigger self-assembly of fibers where the helices are protected from complete unfolding. Our data support an earlier hypothesis that in fibers collagen helices may melt and refold locally when needed, giving fibers their strength and elasticity. Apparently, Nature adjusts collagen hydroxyproline content to ensure that the melting temperature of triple helical monomers is several degrees below rather than above body temperature.T ype I collagen is the most abundant animal protein, and forms the matrix of bone, skin, and other tissues. One would think that matrix proteins should be very stable. Nevertheless, for over half a century it was taken for granted that triple helices of type I collagen melt just several degrees above body temperature (1, 2). Naturally, much attention was paid to the origins (3, 4) and physiological role (5) of this marginal thermal stability. It was proposed, for example, that destabilization of type I collagen by mutations is an important factor in osteogenesis imperfecta, a debilitating and often lethal hereditary disorder characterized by brittle bones (6, 7).Most measurements of collagen denaturation were (and still are) done by scanning at Ϸ0.02-2°C͞min heating rate or after a short equilibration at constant temperature (usually several minutes). To infer the equilibrium melting temperature, T m , one must extrapolate such data to zero heating rate or infinite waiting time (1). But a recent differential scanning calorimetry (DSC) study (8) and our DSC measurements at much slower rates ( Fig. 1) unequivocally show that the apparent T m changes linearly with the logarithm of the heating rate at all rates and equilibration times reported before. Because a logarithmic dependence cannot be extrapolated to zero argument (Fig. 1), the equilibrium T m cannot be inferred from the published data. Either collagen denaturation is an intrinsically nonequilibrium process (8) or the equilibrium T m is lower and the protein is less stable than previously believed.In the present study we demonstrate that the equilibrium T m of collagen does exist, but it is several degrees below body temperature in physiological solution. The thermodynamically preferred conformation of collagen at body temperature is a random coil rather than helix. This must be a deliberate design, because Nature tunes collag...

While the specificity and timing of membrane fusion in diverse physiological reactions, including virus–cell fusion, is determined by proteins, fusion always involves the merger of membrane lipid bilayers. We have isolated a lipid-dependent stage of cell–cell fusion mediated by influenza hemagglutinin and triggered by cell exposure to mildly acidic pH. This stage preceded actual membrane merger and fusion pore formation but was subsequent to a low pH–induced change in hemagglutinin conformation that is required for fusion. A low pH conformation of hemagglutinin was required to achieve this lipid-dependent stage and also, downstream of it, to drive fusion to completion. The lower the pH of the medium applied to trigger fusion and, thus, the more hemagglutinin molecules activated, the less profound was the dependence of fusion on lipids. Membrane-incorporated lipids affected fusion in a manner that correlated with their dynamic molecular shape, a characteristic that determines a lipid monolayer's propensity to bend in different directions. The lipid sensitivity of this stage, i.e., inhibition of fusion by inverted cone–shaped lysophosphatidylcholine and promotion by cone-shaped oleic acid, was consistent with the stalk hypothesis of fusion, suggesting that fusion proteins begin membrane merger by promoting the formation of a bent, lipid-involving, stalk intermediate.