Alfred Ross

Characterization of the linker peptide of the single-chain FV fragment of an antibody by NMR spectroscopy

A comparison of the single‐chain Fv fragment of the antibody McPC603 (scFv) with its corresponding unlinked Fv. fragment has been carried out with 15N‐edited NMR spectroscopy. The two Fv, fragments adopt the same structure, indicating that the linker does not perturb the folding of the domains. This also directly demonstrates that folding in vivo (Fv fragment) and in vitro (scFv. fragment) leads to the same structure. The main differences in the spectra of the uniformly 15N‐labeled scFv and Fv fragments are due to signals of Gly and Ser from the linker peptide of the scF v , fragment. The linker peptide has been mapped with NMR spectra of 15N‐glycine‐ and 15N‐glycine15N‐serine‐labeled scFvfragments. The 15N T2, relaxation data indicate that the linker peptide is more flexible than the rest of the molecule.

Metabolic profiling technologies for biomarker discovery in biomedicine and drug development

The state-of-the-art of nuclear magnetic resonance spectroscopy, mass spectrometry and statistical tools for the acquisition and evaluation of complex multidimensional spectroscopic data in metabolic profiling is reviewed in this article. The continuous evolution of the sensitivity, precision and throughput has made these technologies powerful and extremely robust tools for application in systems biology, pharmaceutical and diagnostics research. Particular emphasis is also given to the collection and storage of biological samples that are subjected to metabolite profiling. Selected examples from preclinical and clinical applications are paradigmatically shown. These illustrate the power of the profiling technologies for characterizing the metabolic phenotype of healthy, diseased and treated subjects. The complexity of disease and drug treatment is asking for an adequate response by integrated and comprehensive metabolite profiling approaches that allow the discovery of new combinations of metabolic biomarkers.

Automation of NMR measurements and data evaluation for systematically screening interactions of small molecules with target proteins.

In this technical note we describe the setup and application of automated sample preparation and usage of flow-through NMR equipment for the characterization of ligand binding on proteins. In addition, we focus on the perspectives of automated analysis of 2D HSQC spectra to identify changes in patterns indicative for ligand binding or changes of sample conditions. In this context we discuss a combination of statistical and non-statistical data analysis.

Secondary structure of human granulocyte colony‐stimulating factor derived from NMR spectroscopy

Recombinant 13N‐. 13C‐labeled human granulocyte colony‐stimulating factor (rh‐metG‐CSF) has been studied by 2D and 3D NMR using uniformly labeled protein as well is residue‐specific 13N‐labeled samples. Assignment of the 1H, 15N backbone, and 60% 1H sidechain resonances has enabled the determination of the secondary structure of the protein. The secondary structure is dominated by α‐helical regions with four stretches of helices between residues 11–41, 71–95, 102–124 and 144–170.

Computer-assisted assignment of multidimensional NMR spectra of proteins: Application to 3D NOESY-HMQC and TOCSY-HMQC spectra

SummaryAn algorithm based on the technique of combinatorial minimization is used for the semi-automated assignment of multidimensional heteronuclear spectra. The program (ALFA) produces the best assignment compatible with the available input data. Even partially misleading or missing data do not seriously corrupt the final assignment. Ambiguous sequences of the possible assignment and all alternatives are indicated. The program can also use additional non-spectroscopic data to assist in the assignment procedure. For example, information from the X-ray structure of the protein and/or information about the secondary structure can be used. The assignment procedure was tested on spectra of mucous trypsin inhibitor, a protein of 107 residues.

Automation of measurements and data evaluation in biomolecular NMR screening

This article reviews the equipment required for biomolecular screening applications in the automated preparation of samples and the acquisition of a large number of NMR data sets. New hardware connecting lab-bench and NMR spectrometers is introduced. In addition, the article focuses on software used for the automated processing of data and the calculation of similarity between spectra - a prerequisite for the identification of test compounds interacting with a target molecule.

Efficient methods for obtaining phase-sensitive gradient-enhanced HMQC spectra

SummaryThree improved versions of the gradient-enhanced HMQC experiment are presented which yield phase-sensitive spectra with increased sensitivity compared to the recently described field-gradient HMQC schemes. The first method uses a complex linear back-prediction in order to generate the FIDs at the t1=0. With this approach, refocusing pulses on the heteronucleus are not necessary. The sequence is especially useful for larger proteins with short relaxation times for the coherences that evolve during t1. In the other two methods lower and shorter gradient pulses or asymmetric gradients are used to optimize sensitivity.

Secondary NOE pathways in 2D NOESY spectra of proteins estimated from homonuclear three-dimensional NOE-NOE nuclear magnetic resonance spectroscopy

Abstract Three-dimensional NOE-NOE intensities are used to estimate second-order τ m contributions to the 2D NOESY intensities. A method to calibrate the 2D and 3D NOE spectra is described which enables comparison of the 2D and 3D intensities. The estimated second-order terms can be used as a direct input into the relaxation-matrix calculations.

Automation of biomolecular NMR screening.

For NMR based screening we review equipment needed for automated preparation of samples and acquisition of a large number of data sets. Hardware connecting lab-bench and NMR spectrometer is described. We focus on software used for automated calculation of the similarity between spectra - a prerequisite for the identification of test compounds interacting with a target-molecule.

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

SummaryThe backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific 15N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.