34 research outputs found
Accurate Structure and Dynamics of the Metal-Site of Paramagnetic Metalloproteins from NMR Parameters Using Natural Bond Orbitals
Novel Entries in a Fungal Biofilm Matrix Encyclopedia
International audienceVirulence of Candida is linked with its ability to form biofilms. Once established, biofilm infections are nearly impossible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we provide a comprehensive analysis of the matrix manufactured by Candida albicans both in vitro and in a clinical niche animal model. We further explore the function of matrix components, including the impact on drug resistance. We uncovered components from each of the macromolecular classes (55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid) in the C. albicans biofilm matrix. Three individual polysaccharides were identified and were suggested to interact physically. Surprisingly, a previously identified polysaccharide of functional importance, β-1,3-glucan, comprised only a small portion of the total matrix carbohydrate. Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched β-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx). Functional matrix proteomic analysis revealed 458 distinct activities. The matrix lipids consisted of neutral glycerolipids (89.1%), polar glycerolipids (10.4%), and sphingolipids (0.5%). Examination of matrix nucleic acid identified DNA, primarily noncoding sequences. Several of the in vitro matrix components, including proteins and each of the polysaccharides, were also present in the matrix of a clinically relevant in vivo biofilm. Nuclear magnetic resonance (NMR) analysis demonstrated interaction of aggregate matrix with the antifungal fluconazole, consistent with a role in drug impedance and contribution of multiple matrix components.IMPORTANCE This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs. The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle
13C and proton NMR studies of horse cytochrome c. Systematic assignment of methyl and methine resonances in both oxidation states
Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes of chymotrypsin
Accurate Structure and Dynamics of the Metal-Site of Paramagnetic Metalloproteins from NMR Parameters Using Natural Bond Orbitals
A natural bond orbital (NBO) analysis of unpaired electron
spin
density in metalloproteins is presented, which allows a fast and robust
calculation of paramagnetic NMR parameters. Approximately 90% of the
unpaired electron spin density occupies metal–ligand NBOs,
allowing the majority of the density to be modeled by only a few NBOs
that reflect the chemical bonding environment. We show that the paramagnetic
relaxation rate of protons can be calculated accurately using only
the metal–ligand NBOs and that these rates are in good agreement
with corresponding rates measured experimentally. This holds, in particular,
for protons of ligand residues where the point-dipole approximation
breaks down. To describe the paramagnetic relaxation of heavy nuclei,
also the electron spin density in the local orbitals must be taken
into account. Geometric distance restraints for <sup>15</sup>N can
be derived from the paramagnetic relaxation enhancement and the Fermi
contact shift when local NBOs are included in the analysis. Thus,
the NBO approach allows us to include experimental paramagnetic NMR
parameters of <sup>15</sup>N nuclei as restraints in a structure optimization
protocol. We performed a molecular dynamics simulation and structure
determination of oxidized rubredoxin using the experimentally obtained
paramagnetic NMR parameters of <sup>15</sup>N. The corresponding structures
obtained are in good agreement with the crystal structure of rubredoxin.
Thus, the NBO approach allows an accurate description of the geometric
structure and the dynamics of metalloproteins, when NMR parameters
are available of nuclei in the immediate vicinity of the metal-site