Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures. The characterization of RNA–protein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X‐ray crystallography, or strategies based on chemical crosslinking, however, can be quite challenging. Herein, we have explored the use of an alternative method, native top‐down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single‐residue level of RNA. Our data show that the electrostatic interactions between HIV‐1 TAR RNA and a peptide comprising the arginine‐rich binding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNA by collisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TAR RNA by top‐down MS. Moreover, the MS data reveal time‐dependent 1:2 and 1:1 stoichiometries of the TAR–tat complexes and suggest structural rearrangements of TAR RNA induced by binding of tat peptide.     

Neural networks to study invariant features of protein folding

Protein secondary structures result both from short-range and long-range interactions. Here neural networks are used to implement a procedure to detect regions of the protein backbone where local interactions have an overwhelming effect in determining the formation of stretches in α-helical conformation. Within the framework of a modular view of protein folding we have argued that these structures correspond to the initiation sites of folding. The hypothesis to be tested in this paper is that sequence identity beside ensuring similarity of the three-dimensional conformation also entails similar folding mechanisms. In particular, we compare the location and sequence variability of the initiation sites extracted from a set of proteins homologous to horse heart cytochrome c. We present evidence that the initiation sites conserve their position in the aligned sequences and exhibit a more reduced variability in the residue composition than the rest of the protein. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 11 November 1998     

Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry

The recognition of the aminoglycosides neomycin and streptomycin by HIV-1 TAR RNA was studied by electrospray ionization mass spectrometry (ESI-MS). Members of the aminoglycoside family of antibiotics are known to target a wide variety of RNA molecules. Neomycin and streptomycin inhibit the formation of the Tat protein–TAR RNA complex, an assembly that is believed to be necessary for HIV replication. The noncovalent complexes formed by the binding of aminoglycosides to TAR RNA and the Tat–TAR complex were detected by ESI-MS. Neomycin has a maximum binding stoichiometry of three and two to TAR RNA and to the Tat–TAR complex, respectively. Data from the ESI-MS experiments suggest that a high affinity binding site of neomycin is located near the three-nucleotide bulge region of TAR RNA. This is consistent with previous solution phase footprinting measurements [H.-Y. Mei et al., Biochemistry 37 (1998) 14204]. Neomycin has a higher affinity toward TAR RNA than streptomycin, as measured by ESI-MS competition binding experiments. A noncovalent complex formed between a small molecule inhibitor of TAR RNA, which has a similar solution binding affinity as the aminoglycosides, and TAR RNA is much less stable than the RNA–aminoglycoside complexes to collisional dissociation in the gas phase. It is believed that the small molecule inhibitor interacts with TAR RNA via hydrophobic interactions, whereas the aminoglycosides bind to RNAs through electrostatic forces. This difference in gas phase stabilities may prove useful for discerning the types of noncovalent forces holding complexes together.     

How possible is the detection of correlated mutations?

The remarkable conservation of protein structure, compared to that of sequences, suggests that, in the course of evolution, residue substitutions which tend to destabilise a particular structure must be compensated by other substitutions that confer greater stability on that structure. Given the compactness of proteins, spatially close residues are expected to undergo the compensatory process. Surprisingly, approaches designed to detect such correlated changes have led, until now, only to limited success in detecting pairs of residues adjacent in the three-dimensional structures. We have undertaken, by simulating the evolution of DNA sequences including sites mutating in a correlated manner, to analyse whether such poor results can be attributed to the detection methods or if this failure could result from a compensatory process more complex than that implicitly underlying the different approaches. Present results show that only methods taking into account the phylogenetic reconstruction can lead to correct detection. Received: 24 April 1998 / Accepted: 8 August 1998 / Published online: 11 November 1998     

Dynamics of the transmembrane domain of the ErbB-2 receptor

The structure and dynamics of the ErbB-2 transmembrane domain have been examined using molecular dynamics techniques both in vacuum and within an explicit hydrated L-α-dilauroyl-phosphatidyl-ethanolamine environment. In-vacuum simulations show that a highly cooperative structural transition occurs frequently within the α-helical transmembrane domain which converts to local π-helices. We show that the α-helix alteration does not depend upon the force field or initial side-chain conformations but is intimately related to the sequence. The membrane-like environment does not prevent the structural transition in the helix but slows down the peptide dynamics indicating that the appearance of a π-bulge is not an artifact of the vacuum approximation. The consequences of π-helix formation could be very huge for the ErbB-2 receptor which is involved in numerous human cancers and also for other membrane proteins wherein similar local structures are also observed experimentally. Received: 9 May 1998 / Accepted: 3 September 1998 / Published online: 17 December 1998     

Sequence dependence of DNA radioprotection by the thiols WR-1065 and WR-151326

Sequence-dependent variations of DNA structure modulate radiation-induced strand breakage. Thiols reduce breakage by scavenging damaging radiolytic OH ^. and repairing sugar radicals. As shown by sequencing gel electrophoresis, WR-1065 radioprotection is modulated by sequence, whereas that of WR-151326, a larger thiol, is more evenly distributed. Molecular modelling was performed on complexes of a 53 bp oligonucleotide (belonging to a natural restriction fragment) with one molecule of WR-1065 or WR-151326. Energy minimised structures exhibit a broadening of the minor groove of an AAATT motif upon WR-1065 binding, and a narrowing of the groove upon WR-151326 binding. Consequently, the accessibility to OH^˙ of H4′ (whose abstraction leads to strand breakage) increases near WR-1065, whereas it decreases near WR-151326. This modifies locally the otherwise homogeneous radioprotection. The effect of WR-151326 strengthens the protection at all tested binding sites, whereas that of WR-1065 diminishes it in some regions, in good agreement with the observed radioprotection distribution. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 11 November 1998     

Choosing GTO basis sets for periodic HF calculations

We investigate numerical linear dependencies of Gaussian-type orbital basis sets employed in the framework of the Hartree-Fock self-consistent field method for periodic structures, which so far have hampered the use of extended basis sets for non-ionic crystals. These linear dependencies occur when diffuse basis functions are included in a basis set in an uncontrolled manner. We use the condition number of the overlap matrix to lead us in the construction of extended basis sets for periodic structures which avoid numerical linear dependencies. Extended basis sets of high quality are optimized for a number of periodic structures (fcc He, α-Be, α-BN, and B1 NaF) with respect to the energy of the constituent atoms or ions. The results obtained with our basis sets, which do not require reoptimization in the crystal environment, compare favorably with those obtained with other extended basis sets reported in the literature. Received: 20 July 1998/Accepted: 21 August 1998 / Published online: 19 October 1998     

RISM-SCF study of the free-energy profile of the Menshutkin-type reaction NH3+CH3Cl→NH3CH3++Cl− in aqueous solution

The free-energy profile for the Menshutkin-type reaction NH₃ + CH₃Cl → NH₃CH₃ ⁺ + Cl⁻ in aqueous solution is studied using the RISM-SCF method. The effect of electron correlation on the free-energy profile is estimated by the RISM-MP2 method at the HF optimized geometries along the reaction coordinate. Solvation was found to have a large influence on the vibrational frequencies at the reactant, transition state and product; these vibrational frequencies are utilized to calculate the zero-point energy correction of the free-energy profile. The computed barrier height and reaction exothermicity are in reasonable agreement with those of experiment and previous calculations. The change of solvation structure along the reaction path is represented by radial distribution functions between solute-solvent atomic sites. The mechanisms of the reaction are discussed from the view points of solute electronic and solvation structures. Received: 26 June 1998/Accepted: 28 August 1998 / Published online: 2 November 1998     

A theoretical study on the first ionic states of vinyl fluoride, vinyl chloride, trifluoroethylene, and trichloroethylene with an analysis of the vibrational structures of the photoelectron spectra

Ab initio calculations have been performed to study the molecular structures and the vibrational levels of the first ionic states of vinyl fluoride, vinyl chloride, trifluoroethylene, and trichloroethylene. The equilibrium molecular structures and vibrational modes of these states are presented. The theoretical ionization intensity curves including the vibrational structures are also presented and compared with the photoelectron spectra. Received: 11 September 1998 / Accepted: 13 October 1998 / Published online: 1 February 1999     

Generalized extended empirical bond-order dependent force fields including nonbond interactions

We present a general approach for describing chemical processes (bond breaking and bond formation) in materials using force fields (FF) that properly describe multiple bonds at small distances while describing nonbond (Coulomb and van der Waals) interactions at long distances. This approach is referred to as the generalized extended empirical bond-order dependent FF. In this paper we use the Brenner empirical bond-order dependent FF for the short-range interactions and report applications on the energetics and structures of graphite crystal, dynamics of molecular crystals, and distortions of bucky tubes. Received: 7 August 1998 / Accepted: 26 October 1998 / Published online: 16 March 1999     

Recent advances in molecular dynamics simulation towards the realistic representation of biomolecules in solution

Coupled advances in empirical force fields and classical molecular dynamics simulation methodologies, combined with the availability of faster computers, has lead to significant progress towards accurately representing the structure and dynamics of biomolecular systems, such as proteins, nucleic acids, and lipids in their native environments. Thanks to these advances, simulation results are moving beyond merely evaluating force fields, displaying expected structural fluctuations, or demonstrating low root-mean-squared deviations from experimental structures and now provide believable structural insight into a variety of processes such as the stabilization of A-DNA in mixed water and ethanol solution or reversible β-peptide folding in methanol. The purpose of this overview is to take stock of these recent advances in biomolecular simulation and point out some common deficiencies exposed in longer simulations. The most significant methodological advances relate to the development of fast methods to properly treat long-range electrostatic interactions, and in this regard the fast Ewald methods are becoming the de facto standard. Received: 9 April 1998 / Accepted: 21 May 1998 / Published online: 13 August 1998     

Thermodynamic behavior of an area-preserving multibaker map with energy

A multibaker map with “kinetic energy” is proposed which incorporates an external field. The map is volume-preserving, time-reversal symmetric and conserves total energy. In an appropriate macroscopic limit, the particle distribution is shown to obey a Smoluchowski-type equation. For the cases without any external field and with a constant external force, the nonequilibrium stationary states are constructed by solving the evolution equation of the partially integrated distribution functions. These states are described by singular functions such as incomplete Takagi functions and Lebesgue's singular functions. In an appropriate macroscopic limit, the mass flows for the stationary states are shown to be identical to the ones expected from the Smoluchowski equation and a “heat flow” proportional to the local energy gradient appears. The Gaspard–Gilbert–Dorfman entropy production is calculated for the stationary states and is shown to be positive. Particularly, for the case with a constant external force, when the energy distribution is independent of the spatial distribution, the entropy production reduces to the one consistent with classical thermodynamics. The result shows that there exists a volume-preserving driven multibaker map whose entropy production is consistent with classical thermodynamics. Received: 17 August 1998 / Accepted: 17 September 1998 / Published online: 15 February 1999     

Prediction of protein structure using a knowledge-based off-lattice united-residue force field and global optimization methods

A united-residue model of polypeptide chains developed in our laboratories with united side-chains and united peptide groups as interaction sites is presented. The model is designed to work in continuous space; hence efficient global-optimization methods can be applied. In this work, we adopted the distance-scaling method that is based on continuous deformation of the original rugged energy hypersurface to obtain a smoothed surface. The method has been applied successfully to predict the structures of simple motifs, such as the three-helix bundle structure of the 10-58 fragment of staphylococcal protein A in de novo folding simulations and more complicated motifs in inverse-folding simulations. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 2 November 1998     

Determination of a set of parameters for the molecular modelling of phosphorothioate DNA

Phosphorothioate DNAs, have emerged as a new class of potent drugs. They are obtained by the replacement of the anionic oxygens of the phophodiester backbone by sulphur. A set of parameters has been developed for the FLEX force field implemented in JUMNA 10.0 to take into account the influence of sulphur on the structure of the DNA double helix. The consistency of our parameters was tested by modelling a phosphorothioate oligomer namely d(GC)8. d(GC)8. Results, obtained on both R-p_S and S-p_S diastereoisomers, were compared to the phosphodiester counterpart and are in agreement with available experimental data. Thus, our set of parameters seems suitable for further molecular modelling of other phosphorothioate oligomers. Received: 15 July 1998 / Accepted: 8 September 1998 / Published online: 10 December 1998     

META 4. Prediction of the metabolism of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment and are often implicated as potential carcinogens. It is generally believed that the carcinogenic potential of polycyclic aromatic hydrocarbons is linked to the formation of ultimate carcinogens generated by metabolic biotransformations. In this paper we propose a methodology that consists of using both quantum chemical properties and structural features of the reaction sites to predict PAH metabolism. Two essential questions have been answered: at which sites will the reaction take place and does the transformation consist of epoxidation or hydroxylation? This methodology has been successfully implemented into an expert system, META, for the evaluation of metabolic transformations of new chemicals. Received: 3 April 1998 / Accepted: 6 July 1998 / Published online: 7 October 1998     

A statistical analytical approach to predict the secondary structure of proteins from amino acid sequence information

A statistical analytical approach has been used to analyze the secondary structure (SS) of amino acids as a function of the sequence of amino acid residues. We have used 306 non-homologous best-resolved protein structures from the Protein Data Bank for the analysis. A sequence region of 32 amino acids on either side of the residue is considered in order to calculate single amino acid propensities, di-amino acid potentials and tri-amino acid potentials. A weighted sum of predictions obtained using these properties is used to suggest a final prediction method. Our method is as good as the best-known SS prediction methods, is the simplest of all the methods, and uses no homologous sequence/family alignment data, yet gives 72% SS prediction accuracy. Since the method did not use many other factors that may increase the prediction accuracy there is scope to achieve greater accuracy using this approach. Received: 4 May 1998 / Accepted: 17 September 1998 / Published online: 10 December 1998     

Examining ligand-protein interactions with binding-energy landscapes

Binding-energy landscapes are used to investigate the thermodynamics of molecular recognition for the pteridine ring, a recognition anchor in binding with dihydrofolate reductase, and two molecules with the same shape but different heteroatom substitutions. The relative importance of hydrogen bonding and hydrophobic interactions in this system is analyzed by comparing these three different decorations of the pteridine scaffold. Received: 5 May 1998 / Accepted: 3 September 1998 / Published online: 17 December 1998     

Conformational constraint as a means for understanding RNA-aminoglycoside specificity

The lack of high RNA target selectivity displayed by aminoglycoside antibiotics results from both their electrostatically driven binding mode and their conformational adaptability. The inherent flexibility around their glycosidic bonds allows them to easily assume a variety of conformations, permitting them to structurally adapt to diverse RNA targets. This structural promiscuity results in the formation of aminoglycoside complexes with diverse RNA targets in which the antibiotics assume distinct conformations. Such differences suggest that covalently linking individual rings in an aminoglycoside could reduce its available conformations, thereby altering target selectivity. To explore this possibility, conformationally constrained neomycin and paromomycin analogues designed to mimic the A-site bound aminoglycoside structure have been synthesized and their affinities to the TAR and A-site, two therapeutically relevant RNA targets, have been evaluated. As per design, this constraint has minimal deleterious effect on binding to the A-site. Surprisingly, however, preorganizing these neomycin-class antibiotics into a TAR-disfavored structure has no deleterious effect on binding to this HIV-1 RNA sequence. We rationalize these observations by suggesting that the A-site and HIV TAR possess inherently different selectivities toward aminoglycosides. The inherent plasticity of the TAR RNA, coupled to the remaining flexibility within the conformationally constrained analogues, makes this RNA site an accommodating target for such polycationic ligands. In contrast, the deeply encapsulating A-site is a more discriminating RNA target. These observations suggest that future design of novel target selective RNA-based therapeutics will have to consider the inherent "structural" selectivity of the RNA target and not only the selectivity patterns displayed by the low molecular weight ligands.     

Anharmonic force field and spectroscopic constants of silene: an ab initio study

High-level ab initio calculations with large basis sets are reported for silene, H₂C=SiH₂. Correlated harmonic force fields are obtained from coupled cluster CCSD(T) calculations with the cc-pVQZ basis (cc-pVTZ for H) while the anharmonic force fields are computed at the MP2/TZ2Pf level. There is excellent agreement with the available experimental data, in particular the equilibrium geometry and the fundamental vibrational frequencies. Many other spectroscopic constants are predicted for the C _{2 v} isotopomers of silene. Received: 27 May 1998 / Accepted: 23 July 1998 / Published online: 9 October 1998     

Information about the biologically relevant properties of Clostridium pasteurianum rubredoxin obtained from modeling and dynamics simulations of molecular variants

Rubredoxins are small electron transfer proteins containing one iron atom at their active site. The rubredoxin from the anaerobic bacterium Clostridium pasteurianum has been subjected to molecular dynamics studies starting from the minimized solvated structure. The results of the simulations have been compared with identical ones carried out with selected mutated forms of the protein obtained by molecular modeling. Surface residues, which are highly conserved among rubredoxins and close to the cysteine ligands, can be replaced by glutamates, i.e. long chain carboxylates. The main structural consequence is a shift of the protein backbone bearing conserved aromatic residues. Reciprocally, substitution of the aromatic residue closest to the iron atom shifts the cysteine-containing peptide fragments. These observations have been related to the changes in electron transfer and redox properties previously measured for this set of rubredoxin molecular variants. Received: 16 May 1998 / Accepted: 4 August 1998 / Published online: 2 November 1998