Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22 degrees C, LDS-P5 formed narrow pores (58 +/- 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 +/- 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures >or=40 degrees C; the process took 4-6 h at 40 degrees C or 35-45 min at 42 degrees C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.     

Significant differences in the activities of alpha-amylases in the absence and presence of polyethylene glycol assayed on eight starches solubilized by two methods

Starch is a reserve chemical source of the energy of the sun found in plants as a water-insoluble granule that differs in their chemical and physical properties, depending on the source. The granules can be solubilized by heating in water or by treatment with various reagents, such as 1M NaOH. alpha-Amylases are widely distributed enzymes that initiate the hydrolysis of starch into low molecular weight maltodextrins. We recently found that the activities of a single alpha-amylase on two different starches were significantly different. We then determined the activities of Bacillus amyloliquefaciens and porcine pancreas alpha-amylases, using eight different starches, solubilized by two methods: autoclaving at 121 degrees C and 1M NaOH at 20 degrees C. There were significant differences in the activities of both of the amylases on all eight of the starches. Previously, it had been found that polyethylene glycol (PEG) stabilized and activated the activities of both enzymes, using a soluble amylose as the substrate. Addition of PEG to the enzymes greatly increased the activities on the eight starches, but the activities still differed significantly. The different activities with the starches were hypothesized as differences in the amounts of secondary and tertiary structures that are partially retained when the different starches are solubilized; the activities on addition of PEG is hypothesized as the formation of highly active species from a series of less active forms.     

SECRET AGENT, an Arabidopsis thaliana O-GlcNAc transferase, modifies the Plum pox virus capsid protein

The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked GlcNAc (O-GlcNAc). While Arabidopsis has two O-GlcNAc transferases, SECRET AGENT (SEC) and SPINDLY (SPY), previous work suggests that SEC modifies PPV-CP and that the modification plays a role in the infection process. Here, we show that when co-expressed in Escherichia coli SEC modifies PPV-CP. Deletion mapping and site-directed mutagenesis identified three threonine and a serine located near the N-terminus of PPV-CP that are modified by SEC. Two of these threonines have recently been shown to be modified in virus from plants suggesting that SEC has the same specificity in plants and E. coli.     

Water stress-sensitized photoinhibition in senescing cotyledons of clusterbean: Changes in thylakoid structures and inactivation of photosystem 2

Seedlings of Cyamopsis tetragonoloba were grown on Petri dishes either in water or water plus 3 % PEG-6000 to induce water stress. The senescing cotyledons experiencing the stress exhibited loss in contents of leaf proteins and chlorophyll (Chl) and a decline in oxygen evolution. The effect of PEG treatment was more pronounced at moderate (MI) than low (LI) irradiance. The stress-induced loss in the activity of superoxide dismutase and increase in the thylakoid lipid peroxidation accompanied a change in the physical status of the bilayer membrane as demonstrated by an enhancement of room temperature Chl a fluorescence polarization and decrease in energy transfer efficiency in pigment assembly. This resulted in a sustained decrease in photosystem 2 activity blocking channels of energy utilization. The absorbed quanta, thus unutilized, were excess even at MI, leading to photoinhibitory response.     

A micellar model system for the role of zeaxanthin in the non-photochemical quenching process of photosynthesis—chlorophyll fluorescence quenching by the xanthophylls

To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2-0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary—an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and β-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and β-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and β-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non-photochemical quenching process in photosynthesis are discussed.     

Phospholipid requirement and pH optimum for the in vitro enzymatic activity of the E. coli P-type ATPase ZntA

Detergent solubilization and purification of the E. coli heavy metal P-type ATPase ZntA yields an enzyme with reduced hydrolytic activity in vitro. Here, it is shown that the in vitro hydrolytic activity of detergent solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. The protein-lipid interaction of ZntA was characterized by enzyme-coupled ATPase assays and fluorescence spectroscopy. Among the most abundant naturally occurring phospholipids, only phosphatidyl-glycerol lipids (PG) enhance the in vitro enzymatic ATPase activity of ZntA. Re-lipidation of detergent purified ZntA with 1,2-dioleoylphosphatidyl-glycerol (DOPG) increases the ATPase activity four-fold compared to the purified state. All other E. coli phospholipids fail to activate the ATPase. Among the phosphatidyl-glycerol family, highest activity was observed for 1,2-dioleoyl-PG followed by 1,2-dimyristoyl-PG, 1,2-dipalmitoyl-PG and 1,2-distearoyl-PG. Increasing intrinsic Trp fluorescence quantum yield upon relipidation of ZntA was used to determine a pH maximum for lipid binding at pH 6.7. The pH dependence of the lipid binding was confirmed by pH-dependent ATPase assays showing maximum activity at pH 6.7. The biophysical characterization of detergent solubilized membrane proteins crucially relies on the conformational stability and functional integrity of the protein under investigation. The present study describes how the E. coli ZntA P-type ATPase can be stabilized and functionally activated in a detergent solubilized system.     

Ethylene glycol and the thermostability of trypsin in a reverse micelle system

The influence of ethylene glycol (EG) on the kinetics of hydrolysis of N-alpha-benzoyl-L-arginine ethyl ether catalyzed by trypsin encapsulated in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT)-based reverse micelles was studied at different temperatures. Ethylene glycol was shown to shift the range of the trypsin activity in the reverse micelles towards higher temperatures. Infrared spectroscopy showed a stabilizing effect of EG on the secondary structure of the protein in the system of reverse micelles. Electron spin resonance spectroscopy showed that the solubilized protein affected the interactions of EG with the polar head groups of AOT and altered the rigidity of the micellar matrix. The results indicate that EG increases the thermostability of the solubilized enzyme in microemulsion media by two mechanisms.     

Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.     

Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S₂ specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.     

Reverse Conformational Changes of the Light Chain-Binding Domain of Myosin V and VI Processive Motor Heads during and after Hydrolysis of ATP by Small-Angle X-Ray Solution Scattering

We used small-angle X-ray solution scattering (SAXS) technique to investigate the nucleotide-mediated conformational changes of the head domains [subfragment 1 (S1)] of myosin V and VI processive motors that govern their directional preference for motility on actin. Recombinant myosin V-S1 with two IQ motifs (MV-S1IQ2) and myosin VI-S1 (MVI-S1) were engineered from Sf9 cells using a baculovirus expression system. The radii of gyration (R_g) of nucleotide-free MV-S1IQ2 and MVI-S1 were 48.6 and 48.8 Å, respectively. In the presence of ATP, the R_g value of MV-S1IQ2 decreased to 46.7 Å, while that of MVI-S1 increased to 51.7 Å, and the maximum chord length of the molecule decreased by ca 9% for MV-S1IQ2 and increased by ca 6% for MVI-S1. These opposite directional changes were consistent with those occurring in S1s with ADP and Vi or AlF₄^− 2 bound (i.e., in states mimicking the ADP/Pi-bound state of ATP hydrolysis). Binding of AMPPNP induced R_g changes of both constructs similar to those in the presence of ATP, suggesting that the timing of the structural changes for their motion on actin is upon binding of ATP (the pre-hydrolysis state) during the ATPase cycle. Binding of ADP to MV-S1IQ2 and MVI-S1 caused their R_g values to drop below those in the nucleotide-free state. Thus, upon the release of Pi, the reverse conformational change could occur, coupling to drive the directional motion on actin. The amount of R_g change upon the release of Pi was ca 6.4 times greater in MVI-S1 than in MV-S1IQ2, relating to the production of the large stroke of the MVI motor during its translocation on actin. Atomic structural models for these S1s based upon the ab initio shape reconstruction from X-ray scattering data were constructed, showing that MVI-S1 has the light-chain-binding domain positioned in the opposite direction to MV-S1IQ2 in both the pre- and post-powerstroke transition. The angular change between the light chain-binding domains of MV-S1IQ2 in the pre- to post-powerstroke transition was ∼ 50°, comparable to that of MII-S1. On the other hand, that of MVI-S1 was ∼ 100° or ∼ 130° much less than the currently postulated changes to allow the maximal stroke size of myosin VI-S1 but still significantly larger than those of other myosins reported so far. The results suggest that some additional alterations or elements are required for MVI-S1 to take maximal working strokes along the actin filament.