Sample convection in liquid-state NMR: why it is always with us, and what we can do about it.

Many NMR experiments on liquids suffer if the sample convects. This is particularly true for applications, such as the measurement of diffusion, that rely on spatial labelling of spins. It is widely assumed that, in most well-conducted experiments with stable temperature regulation, samples do not convect. Unfortunately this is not the case. It is shown here that typical NMR samples show measurable convective flow for all but a very narrow range of temperatures; convection is seen both above and below this range, which can be as small as a degree or so for a mobile solvent such as chloroform. This convection is driven by both vertical and horizontal temperature gradients. Measurements of convection velocity are presented for a range of samples, sample tubes, probes, and temperatures. Both decreasing sample tube inner diameter and changing sample tube material from glass to sapphire can slow convection markedly, with sapphire tubes being particularly effective. Such tubes are likely to be particularly helpful for accurate measurement of diffusion by NMR.

Combined use of filtered and edited 1 H NMR spectroscopy to detect 13 C-enriched compounds in complex mixtures.

In conventional metabolism and pharmacokinetic studies, radioactive isotopes are used to identify and quantify the breakdown products of xenobiotics. However, the stable isotope (13) C provides a cheaper and less hazardous alternative. Metabolites of (13) C-enriched xenobiotics can be detected, quantified and identified by (13) C-filtered NMR spectroscopy. However, one obstacle to using (13) C is its 1.1% natural abundance that produces a background signal in (13) C-filtered NMR spectra of crude biological extracts. The signal makes it difficult to distinguish between (13) C-enriched xenobiotics resonances from endogenous metabolites unrelated to the xenobiotic. This study proposes that the (13) C background signal can be distinguished from resonances of (13) C-enriched xenobiotics by the absence of a (12) C component in the xenobiotic. This is detected by combined analysis of (13) C-filtered and -edited NMR spectra. The theory underlying the approach is described and the method is demonstrated by the detection of sub-microgram amounts of (13) C-enriched phenacetin in crude extracts of hepatocyte microsomes.

Analysis methods for identifying coordinated movements during ligand unbinding.

Molecular dynamics simulations have been applied to unbind biological ligands from their receptors. Conformation changes are observed in the biomolecules during unbinding, but there exists no systematic method to detect these conformation changes. In this work, we have used 'essential dynamics' (ED) and projection to latent structures (PLS) to investigate the conformation changes of the bovine serum retinol-binding protein when retinol unbinds from its receptor site. The results of these analyses characterise a large proportion of the movements that occur during unbinding. We find that the loop regions of retinol-binding protein exhibit the largest movements during unbinding. The sudden changes in unbinding speed during the unbinding process appear not to be caused by sudden changes in protein structure.

Principal components analysis of protein structure ensembles calculated using NMR data.

One important problem when calculating structures of biomolecules from NMR data is distinguishing converged structures from outlier structures. This paper describes how Principal Components Analysis (PCA) has the potential to classify calculated structures automatically, according to correlated structural variation across the population. PCA analysis has the additional advantage that it highlights regions of proteins which are varying across the population. To apply PCA, protein structures have to be reduced in complexity and this paper describes two different representations of protein structures which achieve this. The calculated structures of a 28 amino acid peptide are used to demonstrate the methods. The two different representations of protein structure are shown to give equivalent results, and correct results are obtained even though the ensemble of structures used as an example contains two different protein conformations. The PCA analysis also correctly identifies the structural differences between the two conformations.

Pattern recognition analysis of endogenous cell metabolites for high throughput mode of action identification: removing the postscreening dilemma associated with whole-organism high throughput screening.

Although whole-organism HTS can give clear indications of in vivo activity, typically few clues are given as to the mechanism of action (MOA), and determining the MOA for large numbers of active compounds can be costly and complex-an alternative approach is required. This report demonstrates that it is possible to conduct relatively high throughput MOA characterization of HTS hits utilizing a single sample preparation and analytical method. By monitoring a wide range of endogenous cellular metabolites via (1)H nuclear magnetic resonance spectroscopy, the MOA of herbicides can be predicted using computational methods to compare the metabolite perturbation patterns. Herbicides that induce a characteristic pattern of metabolic perturbation in maize include inhibitors of acetolactate synthase, acetyl co-enzyme A carboxylase, protoporphyrinogen oxidase, 5-enolpyruvylshikimate-3-phosphate synthase, and phytoene desaturase. In soya, photosystem II inhibitors can also be detected, further demonstrating that this method is not limited to inhibitors of enzymes that directly act upon endogenous metabolites, or a single species. The methods, including data analysis, can be readily automated, enabling relatively high throughput MOA elucidation of whole-organism screen hits. Additionally, for compounds with a novel MOA, this approach may lead to MOA identification faster than traditional methods. It is envisaged that application of these data analysis methods to other data types-for example, transcription (mRNA) or translation (protein) profiles-is likely to permit higher throughput with smaller sample requirements, along with ability to discriminate MOAs that are not adequately discriminated based upon endogenous metabolite profiles.

Determination of the NMR structure of the complex between U1A protein and its RNA polyadenylation inhibition element.

RNA-protein recognition is critical to post-transcriptional regulation of gene expression, yet poorly understood at the molecular level. The relatively slow progress in understanding this important area of molecular biology is due to difficulties in obtaining good-quality crystals and derivatives, and in preparing samples suitable for NMR investigation. The determination of the structure of the complex between the human U1A protein and its polyadenylation inhibition element is described here. In this paper, we describe the sample preparation, spectral assignments, construction of the NOE-based distance constraints and methodology for calculating the structure of the complex. The structure was determined to an overall precision of 2.03 A (for all ordered regions), and 1.08 A for the protein-RNA interface. The patterns of hydrogen bonding and hydrophobic interactions at the interface were analysed statistically using the final ensemble of 31 structures.

Anaesthesia for aboriginal Australians.

This prospective study was designed to describe problems that arise when Aboriginal people undergo anaesthesia, in order to develop guidelines for anaesthetists who are not accustomed to treating Aboriginal people. Data were collected on 1122 consecutive different individuals undergoing anaesthesia at Royal Darwin Hospital, 24.5% of whom described themselves as Aboriginal. Aboriginal patients were in a poorer physiological state than were non-Aboriginal patients. The prevalence of diabetes mellitus, renal disease and rheumatic heart disease reported in Aboriginal patients was very high. Communication difficulties were more commonly reported in Aboriginal patients; the most common difficulty was apparent shyness or fear, rather than actual language difficulty. The results suggest that the treatment of Aboriginal people involves diagnosis and management of diverse preoperative medical problems, and that better management may be achieved by learning simple cultural strategies and by adding Aboriginal interpreters and health workers to the anaesthetic team.

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.

Specificity of ribonucleoprotein interaction determined by RNA folding during complex formulation.

Many proteins involved in pre-mRNA processing contain one or more copies of a 70-90-amino-acid alphabeta module called the ribonucleoprotein domain. RNA maturation depends on the specific recognition by ribonucleoproteins of RNA elements within pre-mRNAs and small nuclear RNAs. The human U1A protein binds an RNA hairpin during splicing, and regulates its own expression by binding an internal loop in the 3'-untranslated region of its pre-mRNA, preventing polyadenylation. Here we report the nuclear magnetic resonance structure of the complex between the regulatory element of the U1A 3'-untranslated region (UTR) and the U1A protein RNA-binding domain. Specific intermolecular recognition requires the interaction of the variable loops of the ribonucleoprotein domain with the well-structured helical regions of the RNA. Formation of the complex then orders the flexible RNA single-stranded loop against the protein beta-sheet surface, and reorganizes the carboxy-terminal region of the protein to maximize surface complementarity and functional group recognition.

Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding.

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A protein.

The RNP domain is a very common motif found in hundreds of proteins, including many protein components of the RNA processing machinery. The 70-90 amino acid domain contains two highly conserved stretches of 6-8 amino acids (RNP-1 and RNP-2) in the central strands of a four-stranded antiparallel beta-sheet, packed against two alpha-helices by a conserved hydrophobic core. Using multidimensional heteronuclear NMR, we have mapped intermolecular contacts between the human U1A protein 102 amino acid N-terminal RNP domain and a 31-mer oligonucleotide derived from stem-loop II of U1 snRNA. Chemical shift changes induced on the protein by the RNA define the surface of the beta-sheet as the recognition interface. The reverse face of the protein, with the two alpha-helices, remains exposed to the solvent in the presence of the RNA, and is potentially available for protein-protein contacts in spliceosome assembly or splice site selection. Protein-RNA contacts occur at the single-stranded apical loop of the hairpin, but also in the major groove of the helical stem at neighbouring U.G and U.U non-Watson-Crick base pairs. Examination of a proposed model for the complex in the light of the present results reveals several features of RNA recognition by RNP proteins. The quality of the spectra for this complex of 22 kDa demonstrates the feasibility of NMR investigation of RNA-protein complexes.