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Biomedical subjects

Mark Nitz

Publications and source records attributed to Mark Nitz.

8 recordsLinked to original sources
PubMed2003

Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings.

A protein fusion construct of human ubiquitin with an N-terminal lanthanide binding tag (LBT) enables observation of long-range orientational restraints in solution NMR from residual dipolar couplings (RDCs) due to paramagnetic alignment of the protein. The paramagnetic lanthanide ions Tb3+, Dy3+, and Tm3+ are shown to bind to the LBT and induce different alignment tensors, in agreement with theory. RDCs, measured relative to the diamagnetic Lu3+, range from -7.6 to 5.5 Hz for Tb3+ and -6.6 to 6.1 Hz for Dy3+, while an opposite alignment tensor is observed for Tm3+ (4.5 to -2.9 Hz) at 800 MHz. Experimental RDCs are in excellent agreement with those predicted on the basis of the X-ray structure of the protein.

Humans↗
PubMed2003

Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations.

The development of chemical probes for the investigation of the complex phosphorylation signaling cascades that regulate biological events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biologically active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the enviromentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent maximum emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biologically active ligand through unmasking of the key phosphoserine functionality upon UV irradiation.

14-3-3 Proteins↗
PubMed2003

Lanthanide-binding tags as versatile protein coexpression probes.

Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide-binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long-lived luminescence, excellent X-ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40-fold higher affinity for Tb(3+) ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein-visualization techniques, which include rapid luminescence detection of LBT-tagged proteins in SDS-PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.

Amino Acid Sequence↗
PubMed2003

A powerful combinatorial screen to identify high-affinity terbium(III)-binding peptides.

Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb(3+) ions and intense luminescence properties. This methodology utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissociation constant, K(D)) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb(3+) ions.Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.

Amino Acid Sequence↗
PubMed2001

The unique solution structure and immunochemistry of the Candida albicans beta -1,2-mannopyranan cell wall antigens.

Synthetic oligomers of the antigenic Candida albicans (1-->2)-beta-mannopyranans adopt a compact solution conformation that leads to numerous inter-residue nuclear Overhauser effects, including unprecedented nuclear Overhauser effects between n and n + 3 residues. In excellent agreement with experimentally determined distances, unrestrained molecular dynamics point to a single family of conformations that approximate a compact helical motif with a three-residue repeat for this unique homopolymer. When the synthetic di- to hexasaccharides were employed as inhibitors of monoclonal antibodies, which protect mice against a lethal dose of the yeast pathogen, a novel pattern of inhibitor activity was observed. Instead of the paradigm first reported by Kabat (Kabat, E. A. (1962) Fed. Proc. 21, 694-701; Kabat, E. A. (1966) J. Immunol. 97, 1-11), wherein homo-oligosaccharides exhibit increasing inhibitory activity with increasing size, here the maximum activity is reached for di- and trisaccharides and diminishes significantly for tetra-, penta-, and hexasaccharides. These immunochemical data correlate with the ordered conformation of the beta-1,2-linked mannopyranan and imply that a uniquely small antigenic determinant has potential as a component of synthetic conjugate vaccines against Candida albicans.

Antibodies, Monoclonal↗