597 research outputs found
Crystallization and preliminary X-ray diffraction study of the ligand-binding domain of the bacterial chemotaxis-mediating aspartate receptor of Salmonella typhimurium
Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40
Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-α-L-galactopyranosyl-(1→3)-D-galactose] using various β-agarases. Neoagarobiose hydrolase is an enzyme that acts on the α-1,3 linkage in neoagarobiose to yield D-galactose and 3,6-anhydro-L-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 Å, β = 101.86°. The crystals diffracted to 1.98 Å resolution and possibly contains two molecules in the asymmetric unit
De novo design of a homo-trimeric amantadine-binding protein.
The computational design of a symmetric protein homo-oligomer that binds a symmetry-matched small molecule larger than a metal ion has not yet been achieved. We used de novo protein design to create a homo-trimeric protein that binds the C3 symmetric small molecule drug amantadine with each protein monomer making identical interactions with each face of the small molecule. Solution NMR data show that the protein has regular three-fold symmetry and undergoes localized structural changes upon ligand binding. A high-resolution X-ray structure reveals a close overall match to the design model with the exception of water molecules in the amantadine binding site not included in the Rosetta design calculations, and a neutron structure provides experimental validation of the computationally designed hydrogen-bond networks. Exploration of approaches to generate a small molecule inducible homo-trimerization system based on the design highlight challenges that must be overcome to computationally design such systems
Oblique-incidence reflectometry: one relative profile measurement of diffuse reflectance yields two optical parameters
A new, simple and quick approach, oblique-incidence reflectometry, was used to measure the absorption and reduced scattering coefficients of a semi-infinite turbid medium. An obliquely incident light beam causes the center of the far diffuse reflectance to shift from the point of incidence, where the far diffuse reflectance refers to the diffuse reflectance that is several transport mean free paths away from the incident point. The amount of shift yields the diffusion constant by a simple formula, and the slope of the diffuse reflectance yields the attenuation coefficient. Only the relative profile of the diffuse reflectance is needed to deduce both optical parameters, which makes this method attractive in clinical settings because it does not require a stringent calibration for absolute quantity measurements. This method was tested theoretically by Monte Carlo simulations and experimentally by a reflectometer. Because this method can be used to measure optical properties of biological tissues quickly and requires only inexpensive equipment, it has potential clinical application to the diagnosis of disease or monitoring of treatments
Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain. A study using electrospray ionization mass spectrometry and fluorescence binding studies
Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer
Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening
Pi sampling, derived from the incomplete factorial approach, is an effort to maximize the diversity of macromolecular crystallization conditions and to facilitate the preparation of 96-condition initial screens
Promoting crystallization of antibody–antigen complexes via microseed matrix screening
The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate
A high-throughput colourimetric method for the determination of pH in crystallization screens
The crystallization of proteins is dependent on the careful control of numerous parameters, one of these being pH. The pH of crystallization is generally reported as that of the buffer; however, the true pH has been found to be as many as four pH units away. Measurement of pH with a meter is time-consuming and requires the reformatting of the crystallization solution. To overcome this, a high-throughput method for pH determination of buffered solutions has been developed with results comparable to those of a pH meter
Oblique-incidence reflectometry: one relative profile measurement of diffuse reflectance yields two optical parameters
A new, simple and quick approach, oblique-incidence reflectometry, was used to measure the absorption and reduced scattering coefficients of a semi-infinite turbid medium. An obliquely incident light beam causes the center of the far diffuse reflectance to shift from the point of incidence, where the far diffuse reflectance refers to the diffuse reflectance that is several transport mean free paths away from the incident point. The amount of shift yields the diffusion constant by a simple formula, and the slope of the diffuse reflectance yields the attenuation coefficient. Only the relative profile of the diffuse reflectance is needed to deduce both optical parameters, which makes this method attractive in clinical settings because it does not require a stringent calibration for absolute quantity measurements. This method was tested theoretically by Monte Carlo simulations and experimentally by a reflectometer. Because this method can be used to measure optical properties of biological tissues quickly and requires only inexpensive equipment, it has potential clinical application to the diagnosis of disease or monitoring of treatments
Models of natural mutations including site heterogeneity
New computational models of natural site mutations are developed that account for the different selective pressures acting on different locations in the protein. The number of adjustable parameters is greatly reduced by basing the models on the underlying physical-chemical properties of the amino acids. This allows us to use our method on small data sets built of specific protein types. We demonstrate that with this approach we can represent the evolutionary patterns in HIV envelope proteins far better than with more traditional methods. Proteins 32:289–295, 1998. © 1998 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38528/1/4_ftp.pd
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