José L. Neira

Folding studies on ribonuclease A, a model protein

Ribonuclease A (RNase A), an unusually well defined enzyme, has been a test protein in the study of a wide variety of chemical and physical methods of protein chemistry. These methods have in turn provided many insights into the functional properties of RNase A, as well as topics of general interest in protein biochemistry. The presence of four disulfide bonds and the existence of two cis peptide bonds preceding prolines in the native state have complicated the analysis of the folding pathway of RNase A. In this review, we present some new information about the folding of RNase A obtained recently by quench-flow H/D exchange combined with NMR and single-jump and double-jump stopped-flow techniques.

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

SummaryA method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of1H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the φ and ψ backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His119 side chain in the solution and crystal structures has been detected.

Refined solution structure of bovine pancreatic Ribonuclease A by1H NMR methods. Sidechain dynamics

An extension of the assignment of the1H NMR spectra of bovine pancreatic Ribonuclease A has been carried out at 600 MHz covering all sidechain protons, including those belonging to long sidechain residues. Vicinal coupling constants,3JHNα and3Jαβ and3Jαβ′ have been measured with the purpose of imposing torsional constraints on the backbone ϕ angle as well as to introduceHββ′ specific assignments. On the basis of distance constraints evaluated from 1285 assigned NOEs, a first restrained molecular dynamics calculation has been carried out. The resulting root-mean-square deviation for the backbone atoms is 1.5 Å as compared to 1.8 Å for the 360 MHz NOE basis. For heavy atoms in sidechains, these numbers were, respectively, 3.0 and 2.5 Å A second calculation was carried out by introducing 37 dihedral angle constraints on backbone ϕ, torsions and five onX1 angular torsions as well as with stereospecific assignments for nearly 50 residues. Backbone and sidechain RMS deviations were, respectively, 1.2 and 2.0 Å. Sidechains have been classified in terms of their dynamical behavior aroundX1 after considering jointly the coupling information and that resulting from the second set of calculated structures. Results are discussed in relation to the crystal structure.

Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites

The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24+/-0.15. The variations of free energy in the absence of denaturant [DeltaG(H(2)O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15+/-2.3 kJ.mol(-1) and 19.87+/-0.71 kJ.mol(-1).M(-1) for the dissociation of the native dimer and 14.77+/-1.63 kJ.mol(-1) and 5.23+/-0.21 kJ.mol(-1).M(-1) for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69 kJ.mol(-1) and 30.33 kJ.mol(-1).M(-1) respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established.

p8 and prothymosin alpha : Unity is strength

p8 and prothymosine a are two natively unstructured proteins with anti-apoptotic activity. We showed that their interaction results in the formation of a one-to-one heterodimer complex with stable structure. To test whether the heterodimer bears the function previously attributed to both proteins, we monitored the consequences on apoptosis of modulating in vitro the concentrations of both proteins. Over-expression was obtained by transfection of appropriate vectors and inhibition by using specific siRNAs. In all conditions inhibition of apoptosis correlated with the level of the partner with lowest concentration, demonstrating that the anti-apoptotic effect previously attributed to each proteins was in fact borne by the p8/ProTa complex, the two proteins, being individually inactive. These results show that the function attributed to a natively unfolded protein might actually belong to a multi-protein complex in which the protein of interest is engaged.

NMR for chemists and biologists

1. The basis of Nuclear Magnetic Resonance Spectroscopy 1.1. Introduction 1.2. Physical principles of NMR spectroscopy 1.2.1. The basis of NMR spectroscopy: a vector approach 1.2.2. The basis of NMR spectroscopy: a naive quantum approach 1.2.3. The nuclei in NMR 1.3. Spin relaxation 1.3.1. Spin-lattice relaxation time 1.3.2. Spin-spin relaxation time 1.3.3. Sources of variation in local fields 1.4. Pulse techniques 1.4.1. How a pulse works: the time and frequency domains 1.4.2. Multipulse experiments: measurement of the T1- and T2-relaxation times as examples 1.5. Practical aspects of NMR 1.5.1. The magnet 1.5.2. The probe 1.5.3. The lock-system 1.5.4. The transmitter/receiver system: quadrature detection 1.5.5. The shim system 1.5.6. Pulse field gradients 1.5.7. Sample preparation 1.6. References 2. Spectroscopic parameters in Nuclear Magnetic Resonance 2.1. The chemical shift and the spectral intensity 2.1.1. The shielding screening constant 2.1.2. The chemical shift 2.1.3. Signal intensity 2.2. The scalar coupling constant 2.2.1. Spin-spin coupling2.2.2. How does spin-spin coupling occur? 2.2.3. Variations in the value of J 2.2.4. Spin-spin decoupling 2.3. The nuclear Overhauser effect 2.3.1. The basis of the NOE: a two spin system 2.3.2. NOEs in multi-spin systems 2.4. References 3. Basic NMR experiments 3.1. Introduction 3.2. 1D NMR 3.2.1. Sensitivity and frequency 3.2.2. Acquisition and processing 3.2.3. 1D spectra of 1H, 13C,31P and 19F 3.3. Multidimensional NMR 3.3.1. Generating dimensions in NMR 3.3.2. 2D data acquisition and processing 3.4. Homonuclear shift correlation: correlations through the chemical bond 3.4.1. COSY. Experiment interpretation and practical aspects 3.4.2. TOCSY. Practical aspects 3.4.3. Correlation for diluted spins: the INADEQUATE experiment. Double-quantum selection 3.5. Heteronuclear shift correlation: correlations through the chemical bond 3.5.1. Polarization transfer experiments: SPT and INEPT sequences indirect spectroscopy 3.5.2. Heteronuclear single bond correlations: HSQC and HMQC 3.5.3. Double resonance experiments: homonuclear spin decoupling heteronuclear double resonance and broadband decoupling 3.6. Correlations through space 3.6.1. Steady-state NOE 3.6.2. Kinetic or transient NOE 3.6.3. The 2D-NOESY sequence and practical aspects of the experiment 3.7. References 4. Biomolecular NMR 4.1. Introduction 4.2. Why biomolecules? Main applications 4.3. Structure of biomolecules 4.3.1. Homonuclear and heteronuclear (triple resonance) assignment in proteins 4.3.2. Nucleic acids 4.4. Biomolecular dynamics 4.4.1. Comparison with other spectroscopies and other structural biophysical techniques 4.4.2. Movements in the ps-s range 4.5. Biomolecular interactions (NMR in drug discovery) 4.5.1. Order of the affinities measured 4.5.2. Experiments in NMR screening 4.6. Other applications 4.6.1. Metabolomics 4.6.2. Solid state NMR and HR-MAS 4.7. References

Turbulent times in southern European science

1 Akbarnia BA, Mundis GM Jr, Salari P, Yaszay B, Pawelek JB. Innovation in growing rod technique: a study of safety and effi cacy of a magnetically controlled growing rod in a porcine model. Spine 2012; 37: 1109–14. direct costs were estimated at €41 406 (

Falling down: landscape and kinetics of one-dimensional protein folding.

54 653) per patient. Considering the current price of the rods, health-care costs for implantation, and medical visits for magnetic distractions, the management of early-onset scoliosis with magnetically controlled growing rods should not exceed €35 000 (

The Bronze Age of science in Spain

46 037) during a similar follow-up period. Therefore, if the eff ectiveness of magnetically controlled growing rods is further confi rmed by multicentre trials, this could provide a preferential situation in which a breakthrough technology proves cost-saving.

Spain should implement a model that's known to work.

In this issue of Structure, Tsytlonok and colleagues describe the folding landscape of the giant HEAT-repeat protein PR65/A (a molecular adaptor of protein phosphatase 2A) by using experimental and theoretical methods. Both approaches agree in suggesting the presence of parallel folding pathways with several intermediates.