Measurement of Dipolar Cross-Correlation in Methylene Groups in Uniformly 13C-, 15N-Labeled Proteins

A CC(CO)NH TOCSY-based 3D pulse scheme is presented for measuring (1)H-(13)C dipole-dipole cross-correlated relaxation at CH(2) positions in uniformly (13)C-, (15)N-labeled proteins. Simulations based on magnetization evolution under relaxation and scalar coupling interactions show that cross-correlation rates between (1)H-(13)C dipoles in CH(2) groups can be simply obtained from the intensities of (13)C triplets. The normalized cross-correlation relaxation rates are related to cross-correlation order parameters for macromolecules undergoing isotropic motion, which reflect the degrees of spatial restriction of CH(2) groups. The study on human intestinal fatty acid binding protein (131 residues) in the presence of oleic acid demonstrates that side chain dynamics at most CH(2) positions can be characterized for proteins less than 15 kDa in size, with the proposed TOCSY-based approach.     

Dynamics of β-CH and β-CH2 Groups of Amino Acid Side Chains in Proteins

The dynamics of amino acid side chains of uniformly 13C/15N-enriched ribonuclease T1 (RNase T1) have been investigated. Heteronuclear longitudinal relaxation rates, 1H/13C NOEs, and transverse cross-correlated cross-relaxation rates between the Sx and the SxIz1Iz2 operators (SIIS cross relaxation) [Ernst and Ernst (1994) J. Magn. Reson., A110, 202-213] have been determined in this study. New pulse sequences for measuring the longitudinal relaxation time and the heteronuclear NOE of aliphatic side chain carbon nuclei were developed using the CCONH type of magnetization transfer and 1HN detection. In addition, an improved pulse sequence for the determination of the SIIS cross relaxation is presented. For the analysis of the relaxation rates, the model of restricted rotational diffusion around the 1 dihedral angle has been applied [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. These techniques were used in order to describe the side chain dynamics of the small globular protein RNase T1 (104 amino acids, MW about 11 kDa). Qualitative values of microdynamical parameters were obtained for 73 out of 85 amino acid side chains (glycine and alanine residues excepted) whereas more quantitative values were derived for 67 -CH and -CH2 groups.     

Vitamin D: structure-function analyses and the design of analogs.

There is continuing and emerging new interest in the development of vitamin D analogs resulting from the recognition that analogs of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] may be therapeutically useful. Side chain analogs of this steroid hormone are of particular interest because a family of lead structures have recently emerged for possible use in the treatment of certain types of cancers and skin diseases. Because of the chaotic array of side chain structures which exhibit useful therapeutic indices for these purposes, a more systematic approach towards developing intelligible structure-function information needs development. Accordingly, a method has been devised to analyze analogs as to their side chain topology based on identifying specific occupancy volumes through conformational analysis. Dot maps have been constructed as an indication of the volume in space which the side chain of 1 alpha,25-(OH)2-D3 or analogs is permitted to occupy. Volume exclusion analyses based on comparison of structural and biological data for 1 alpha,25-(OH)2-D3 and analogs are anticipated to lead to a more cogent model for drug design. A cautionary note on the limitations of this approach is discussed.     

Exploring the conformational space of protein side chains using dead-end elimination and the A* algorithm

We describe an algorithm which enables us to search the conformational space of the side chains of a protein to identify the global minimum energy combination of side chain conformations as well as all other conformations within a specified energy cutoff of the global energy minimum. The program is used to explore the side chain conformational energy surface of a number of proteins, to investigate how this surface varies with the energy model used to describe the interactions within the system and the rotamer library. Enumeration of the rotamer combinations enables us to directly evaluate the partition function, and thus calculate the side chain contribution to the conformational entropy of the folded protein. An investigation of these conformations and the relationships between them shows that most of the conformations near to the global energy minimum arise from changes in side chain conformations that are essentially independent; very few result from a concerted change in conformation of two or more residues. Some of the limitations of the approach are discussed. Proteins 33:227–239, 1998. © 1998 Wiley-Liss, Inc.     

Design of a structural motif ‘dendroid’ to mimic protein: Protein interactions via intramolecular self-organisation

Summary The design and synthesis of a structural motif which can mimic protein-protein interactions is described. These moieties, termed asdendroids (Greekdendron, a tree), are low-molecular-weight structures which are based on self-organising dendritic polymers.     

A general Bayesian method for an automated signal class recognition in 2D NMR spectra combined with a multivariate discriminant analysis

Summary A generally applicable method for the automated classification of 2D NMR peaks has been developed, based on a Bayesian approach coupled to a multivariate linear discriminant analysis of the data. The method can separate true NMR signals from noise signals, solvent stripes and artefact signals. The analysis relies on the assumption that the different signal classes have different distributions of specific properties such as line shapes, line widths and intensities. As to be expected, the correlation network of the distributions of the selected properties affects the choice of the discriminant function and the final selection of signal properties. The classification rule for the signal classes was deduced from Bayes's theorem. The method was successfully tested on a NOESY spectrum of HPr protein from Staphylococcus aureus. The calculated probabilities for the different signal class memberships are realistic and reliable, with a high efficiency of discrimination between peaks that are true NOE signals and those that are not.     

Partial NMR assignments for uniformly (13C, 15N)-enriched BPTI in the solid state

We demonstrate that high-resolution multidimensional solid state NMR methods can be used to correlate many backbone and side chain chemical shifts for hydrated micro-crystalline U-¹³C,¹⁵N Basic Pancreatic Trypsin Inhibitor (BPTI), using a field strength of 800 MHz for protons, magic angle sample spinning rates of 20 kHz and proton decoupling field strengths of 140 kHz. Results from two homonuclear transfer methods, radio frequency driven dipolar recoupling and spin diffusion, were compared. Typical ¹³C peak line widths are 0.5 ppm, resulting in C-C and C-CO regions that exhibit many resolved peaks. Two-dimensional carbon–carbon correlation spectra of BPTI have sufficient resolution to identify and correlate many of the spin systems associated with the amino acids. As a result, we have been able to assign a large number of the spin systems in this protein. The agreement between shifts measured in the solid state and those in solution is typically very good, although some shifts near the ion binding sites differ by at least 1.5 ppm. These studies were conducted with approximately 0.2 to 0.4 mol of enriched material; the sensitivity of this method is apparently adequate for other biological systems as well.