Protein NMR spectra

Selective labelling of the two diastereotopic methyl groups of i-leucine (144) has enabled their fates during secondary metabolic reactions to be elucidated [66]. Moreover, in the context of protein interactions, differentiation of the leucine pro-R and pro-S methyl groups in protein NMR spectra allows molecular recognition phenomena to be studied [67]. Recently, efficient routes to both forms of Relabeled leucine, based on application of an auxiliary-controlled stereoselective conjugate addition reaction (Scheme 6.27) have been described [68]. Thus, starting... 

HCA(CO)N experiments. These names are deliberately descriptive and should suggest the correlations that these experiments are intended to establish by analogy with the HNCA experiment described here. All these correlation experiments acting in concert are designed to ensure optimal and mostly complete, unique and unambiguous assignments of Tf-resonance peaks to nuclei in given protein NMR spectra. This level of information is usually sufficient... 

Nuclear Magnetic Resonance (NMR) spectroscopy is one of the most important tools in structural studies of chemical compounds, ranging from small molecules up to medium-sized proteins. NMR spectra provide valuable information about molecular structure, interactions and dynamics. However, there is stiU a need for more robust and more effective methods of acquisition and processing of NMR data. 

Shimba N, Stem AS, Craik CS, Hoch JC, Dotsch V (2003) Elimination of Calpha splitting in protein NMR spectra by deconvolution with maximum entropy reconstmction. J Am Chem Soc 125 2382-2383... 

Malmodin D, Billeter M (2005) High-throughput analysis of protein NMR spectra. Prog Nucl Magn Reson Spectrosc 46(2-3) 109-129... 

Nguyen TH, Ozawa K, Stanton-Cook M et al (2010) Generation of pseudocontact shifts in protein NMR spectra with a genetically encoded cobalt(ll)-binding amino acid. Angew Chem Int Ed Engl 50(3) 692-694... 

The issue of what causes line broadening in solid-state NMR was thoroughly investigated by Su et al To understand the linewidth contributions to membrane protein NMR spectra, T2 relaxation times of uniformly labeled residues which show that the homogeneous line widths are determined by conformation-independent factors, including residual dipolar coupling, J-coupling, and intrinsic T2 relaxation. Examples of this were shown by TAT and other peptides. 

Combined with a difference technique the method results in substantial simplifications of the complex protein nmr spectra and in an effective increase in the spectral resolution for the polarized groups. This permits detailed studies of various interactions of proteins, such as the enzyme-inhibitor interactions discussed in sections 5,2 and 6.2 for RNase A and lysozyme. For both enzymes the photo-CIDNP spectrum proved to be very sensitive to the presence of inhibitors, albeit in conpletely different ways Examples of applications to the study of protein-nucleic acid interactions will be treated by Hilbers et al. elsewhere in this volume. We believe that in spite of its brief existence the photo-CIDNP method is potentially a powerful tool for the study of protein structure in solution. 

VR, the inputs correspond to the value of the various parameters and the network is 1 to reproduce the experimentally determined activities. Once trained, the activity of mown compound can be predicted by presenting the network with the relevant eter values. Some encouraging results have been reported using neural networks, have also been applied to a wide range of problems such as predicting the secondary ire of proteins and interpreting NMR spectra. One of their main advantages is an to incorporate non-linearity into the model. However, they do present some problems Hack et al. 1994] for example, if there are too few data values then the network may memorise the data and have no predictive capability. Moreover, it is difficult to the importance of the individual terms, and the networks can require a considerable 1 train. 

The Novosibirsk Institute of Organic Chemistry has developed a method for computer-aided retrieval of stmctural information from H-nmr using its database of 50,000 spectra (72). Eraser WUHams Ltd. (Scientific Systems) has special software to search its E-nmr database (73). Protein nmr data have been compiled into a relational database at the University of Wisconsin (74). 

A gene encoding this sequence was synthesized and the corresponding protein, called Janus, was expressed, purified, and characterized. The atomic structure of this protein has not been determined at the time of writing but circular dichroic and NMR spectra show very clear differences from B1 and equally clear similarities to Rop. The protein is a dimer in solution like Rop and thermodynamic data indicate that it is a stably folded protein and not a molten globule fold like several other designed proteins. 

Figure 18.16 One-dlmenslonal NMR spectra, (a) H-NMR spectrum of ethanol. The NMR signals (chemical shifts) for all the hydrogen atoms In this small molecule are clearly separated from each other. In this spectrum the signal from the CH3 protons Is split Into three peaks and that from the CH2 protons Into four peaks close to each other, due to the experimental conditions, (b) H-NMR spectrum of a small protein, the C-terminal domain of a cellulase, comprising 36 amino acid residues. The NMR signals from many individual hydrogen atoms overlap and peaks are obtained that comprise signals from many hydrogen atoms. (Courtesy of Per Kraulis, Uppsala, from data published in Kraulis et al.. Biochemistry 28 7241-7257, 1989.)...

Two-dimensional NMR spectra of proteins are interpreted by the method of sequential assignment... 

NMR spectra have been reported for the Rieske-type ferredoxins from Xanthobacter strain Py2 (88) and of toluene 4-monooxygenase from Pseudomonas mendocina (T4MOC) (88a) as well as for the water-soluble Rieske fragment from the bci complex of Paracoccus deni-trificans (ISFpd) (89). The spectra of these proteins are similar, which is consistent with the close structural relationship between the three proteins. In the reduced (paramagnetic) state, all three proteins show several hyperfine-shifted resonances between +83 and -16 ppm at 400 MHz or between 110 and +25 ppm at 300 MHz (Table X). 

---