We studied the molecular arrangement of two-dimensional streptavidin crystals at the air-water interface over a range of pH values. We quantified the varying amounts of coexisting P1, P2, and C222 crystals in the different morphologies observed at pH 4.5-6.5. Chiral, needlelike crystals at pH 4.5 consist of P1 crystals with frequent line defects. Larger chiral domains near pH 5 are essentially all P1 coexisting with a small amount of P2, whereas at slightly higher pH values (near pH 5.5), H-shaped domains contain 4 times as much P1 coexisting with a P2/C222 mixture. Morphologies intermediate to these shapes exhibit intermediate compositions. Between pH ∼6-7, crystals all display a characteristic dendritic-X morphology, but arrangement at the molecular level is quite different compared with lower pH values. Crystals are mostly P2 in symmetry near pH 6, but at pH 7 and above, crystals have C222 symmetry. Coexistence of P2 and C222 crystals occurs at intermediate pH values. We determined the orientation and arrangement of streptavidin molecules in P1, P2, and C222 crystals relative to the directions exhibiting faster growth. The direction of faster growth in P1 crystals includes both interactions between biotin-free subunits and interactions between biotin-bound subunits. In the P2 arrangement, growth in the direction of intermolecular biotin-free subunits is preferred, whereas growth is faster along the biotin-bound direction of C222 crystals. We developed a model of the molecular arrangement for the observed solid-phase coexistence in these crystals.
To obtain a general understanding of the effect of intermolecular interactions on the mechanisms of two-dimensional protein crystallization, we grow protein crystals and elicit a bulk molecular manipulation by changing system pH. Two-dimensional crystals of the bacterial protein streptavidin grown on a biotinylated lipid monolayer at an air-water interface, in the presence of the noncrystallizable impurity avidin, exhibit crystallographic and morphological changes as a function of subphase pH. Large twodimensional crystalline arrays form within minutes across a pH range from 1.5 to 11. Crystals exhibit different pH-dependent structures, lattices with P1 symmetry for 1.5 < pH < 5, P1 and P2 lattices for 5 < pH < 6, and C222 lattices for 7 < pH < 11. P1 crystals nucleate rapidly and form thin needle-shaped crystals consistent with a strong growth anisotropy between the two crystallographic growth directions. C222 crystals grow more isotropically and exhibit H-and X-shapes. The nucleation rates and aspect ratios of C222 crystals are also pH-dependent, both properties increasing with increasing pH. The transition from C222 to P1 or P2 crystals can be accomplished in minutes by lowering the system pH. The reverse transition, however, does not occur subsequent to a corresponding increase in system pH. Instead, new C222 crystals form, but no reconfiguration of existing crystals is observed.
We describe an optical technique for the measurement of macromolecular adsorption at the solid/liquid interface when multiple species are present. The technique combines surface plasmon resonance (SPR) with simultaneous surface plasmon enhanced fluorescence (SPEF). The relative ease of construction and linear correlation between SPR and SPEF signals make the technique amenable for coadsorption studies or multiple ligand binding experiments. Here, we demonstrate the utility of the technique with a biotin/ avidin/BSA "sandwich" experiment. We then apply SPR/SPEF for the simultaneous monitoring of enzyme adsorption and substrate cleavage of a protease interacting with a substrate surface.
We describe the adsorption and catalytic behavior of the serine protease subtilisin BPN′ on controlled pore glass (CPG) beads with a short (aminopropyl) or a long (aminoalkyl CH2 > 12) chain covalent link separating the reporter peptide succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPFpNA) from the surface. The propyl-linked sAAPFpNA modified glass surface (aminopropyl CPG:sAAPFpNA) showed a 2-fold increase in protease adsorption over an aminopropyl-glass surface. In contrast, the sAAPFpNA surface with the long chain connector showed a 2-fold drop in adsorption relative to an aminoalkyl surface. BPN′-catalyzed hydrolysis rates showed an inverse relationship to adsorption. Water-soluble polymers [poly(vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), poly(4-vinylpyridine-N-oxide) (PVPO) and a copolymer of 1-vinyl-2-pyrrolidone and 1-vinylimidazole (PVPVI)] neutralize the 2-fold increase in BPN′ adsorption and provide more than a 3-fold increase in the initial rate of hydrolysis for BPN′-catalyzed cleavage of pNA. Another water-soluble polymer, poly(vinyl alcohol) (PVA), causes only a slight adsorption decrease and hydrolysis increase for the BPN′, aminopropyl CPG:sAAPFpNA system. None of the polymers causes a significant change in BPN′-catalyzed hydrolysis of, or adsorption on, aminoalkyl (CH2 > 12) CPG:sAAPFpNA. The apparent mechanism behind these effects is one in which the long alkyl chains and adsorbed polymers decrease the amount of adsorbed enzyme and increase the amount available for reaction in solution. A model is presented which describes the relationship between adsorption and surface hydrolysis. Materials and MethodsEnzyme Solution. The steps used to isolate, purify, and prepare a stock solution of the serine protease used here, subtilisin BPN′ (MW ) 27 534 g/mol), from Bacillus amyloliquefaciens are outlined elsewhere. 14 Prior to use, the enzyme stock solution is
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