Evolutionary Landscapes for the Acquisition of New Ligand Recognition by RNA Aptamers

The evolution of ligand specificity underlies many important problems in biology, from the appearance of drug resistant pathogens to the re-engineering of substrate specificity in enzymes. In studying biomolecules, however, the contributions of macromolecular sequence to binding specificity can be obscured by other selection pressures critical to bioactivity. Evolution of ligand specificity in vitro—unconstrained by confounding biological factors—is addressed here using variants of three flavin-binding RNA aptamers. Mutagenized pools based on the three aptamers were combined and allowed to compete during in vitro selection for GMP-binding activity. The sequences of the resulting selection isolates were diverse, even though most were derived from the same flavin-binding parent. Individual GMP aptamers differed from the parental flavin aptamers by 7 to 26 mutations (20 to 57% overall change). Acquisition of GMP recognition coincided with the loss of FAD (flavin-adenine dinucleotide) recognition in all isolates, despite the absence of a counter-selection to remove FAD-binding RNAs. To examine more precisely the proximity of these two activities within a defined sequence space, the complete set of all intermediate sequences between an FAD-binding aptamer and a GMP-binding aptamer were synthesized and assayed for activity. For this set of sequences, we observe a portion of a neutral network for FAD-binding function separated from GMP-binding function by a distance of three mutations. Furthermore, enzymatic probing of these aptamers revealed gross structural remodeling of the RNA coincident with the switch in ligand recognition. The capacity for neutral drift along an FAD-binding network in such close approach to RNAs with GMP-binding activity illustrates the degree of phenotypic buffering available to a set of closely related RNA sequences—defined as the sets functional tolerance for point mutations—and supports neutral evolutionary theory by demonstrating the facility with which a new phenotype becomes accessible as that buffering threshold is crossed.     

Glutamate counteracts the denaturing effect of urea through its effect on the denatured state

The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 m potassium l-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of l-glutamate, suggesting that l-glutamate has the ability to counteract the denaturing effect of urea. d-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of l-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 m potassium l-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N lambda-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.     

Predicted molecular structure of the mammalian cell entry protein Mce1A of Mycobacterium tuberculosis

The proposed role of the mammalian cell entry protein 1A (Mce1A) of Mycobacterium tuberculosis is to facilitate invasion of host cells. The structure of Mce1A was modelled on the basis of the crystal structure of Colicin N of Escherichia coli by fold prediction and threading. Mce1A, as the model predicts, is an alpha/beta protein consisting of two major (alpha and beta) domains, connected by a long alpha helix. The model further revealed that the protein contains 12 helices, 9 strands, and 1 turn. The final model of Mce1A was verified through the program VERIFY 3D and more than 90% of the residues were in the favourable region. A mouse monoclonal antibody, TB1-5 76C, is directed to an epitope within a 60-mer peptide that has been shown to promote uptake of bacteria in mammalian cells. We show here that the epitope could be narrowed down to a core of 4 amino acids, TPKD. Upstream flanking residues, KRR also contributed to binding. Mce2A does not promote uptake in mammalian cells and sequence comparison of Mce1A and Mce2A indicates that the epitope mediates uptake. The epitope was located at the surface of the Mce1A model at the distal beta strand-loop region in the beta domain. The localization of this epitope in the model confirms its potential role in promoting uptake of M. tuberculosis in host cells.     

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.     

Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E,isolated from the blood samples of beta-thalassemic patients

Hemoglobin A(2) (alpha(2)delta(2)), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is a failure of beta-chain production, HbA(2) acts as the predominant oxygen delivery mechanism. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of beta globin gene, when combines with beta-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA(2) and HbE are reported here. HbA(2) and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from beta-thalassemia minor and E beta-thalassemia. X-ray diffraction data of HbA(2) and HbE were collected upto 2.1 and 1.73 A, respectively. HbA(2) crystallized in space group P2(1) with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and beta=99.80 degrees whereas HbE crystallized in space group P2(1)2(1)2(1) with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R(2) state.     

Dynamics of DNA molecules in a membrane channel probed by active control techniques

The dynamics of single-stranded DNA in an alpha-Hemolysin protein pore was studied at the single-molecule level. The escape time for DNA molecules initially drawn into the pore was measured in the absence of an externally applied electric field. These measurements revealed two well-separated timescales, one of which is surprisingly long (on the order of milliseconds). We characterized the long timescale as being associated with the binding and unbinding of DNA from the pore. We have also found that a transmembrane potential as small as 20 mV strongly biased the escape of DNA from the pore. These experiments have been made possible due to the development of a feedback control system, allowing the rapid modulation of the applied force on individual DNA molecules while inside the pore.     

Increased bending rigidity of single DNA molecules by H-NS,a temperature and osmolarity sensor

Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing. It plays a key role in cell response to changes in temperature and osmolarity. Force-extension measurements of single, twist-relaxed lambda-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates. Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15-20 bp. These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity. These effects are not observed above 32 degrees C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing. Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell.     

Interactions of hydrophobic peptides with lipid bilayers: Monte Carlo simulations with M2delta

We introduce here a novel Monte Carlo simulation method for studying the interactions of hydrophobic peptides with lipid membranes. Each of the peptide's amino acids is represented as two interaction sites: one corresponding to the backbone alpha-carbon and the other to the side chain, with the membrane represented as a hydrophobic profile. Peptide conformations and locations in the membrane and changes in the membrane width are sampled using the Metropolis criterion, taking into account the underlying energetics. Using this method we investigate the interactions between the hydrophobic peptide M2delta and a model membrane. The simulations show that starting from an extended conformation in the aqueous phase, the peptide first adsorbs onto the membrane surface, while acquiring an ordered helical structure. This is followed by formation of a helical-hairpin and insertion into the membrane. The observed path is in agreement with contemporary understanding of peptide insertion into biological membranes. Two stable orientations of membrane-associated M2delta were obtained: transmembrane (TM) and surface, and the value of the water-to-membrane transfer free energy of each of them is in agreement with calculations and measurements on similar cases. M2delta is most stable in the TM orientation, where it assumes a helical conformation with a tilt of 14 degrees between the helix principal axis and the membrane normal. The peptide conformation agrees well with the experimental data; average root-mean-square deviations of 2.1 A compared to nuclear magnetic resonance structures obtained in detergent micelles and supported lipid bilayers. The average orientation of the peptide in the membrane in the most stable configurations reported here, and in particular the value of the tilt angle, are in excellent agreement with the ones calculated using the continuum-solvent model and the ones observed in the nuclear magnetic resonance studies. This suggests that the method may be used to predict the three-dimensional structure of TM peptides.     

Insights into the relation between mRNA and protein expression patterns: I. Theoretical considerations

Translation is a central cellular process in every organism and understanding translation from the systems (genome-wide) perspective is very important for medical and biochemical engineering applications. Moreover, recent advances in cell-wide monitoring tools for both mRNA and protein levels have necessitated the development of such a model to identify parameters and conditions that influence the mapping between mRNA and protein expression. Experimental studies show a lack of correspondence between mRNA and protein expression profiles. In this study, we describe a mechanistic genome-wide model for translation that provides mapping between changes in mRNA levels and changes in protein levels. We use our model to study the system in detail and identify the key parameters that affect this mapping. Our results show that the correlation between mRNA and protein levels is a function of both the kinetic parameters and concentration of ribosomes at the reference state. In particular, changes in concentration of free and total ribosomes in response to a perturbation; changes in initiation and elongation kinetics due to competition for aminoacyl tRNAs; changes in termination kinetics; average changes in mRNA levels in response to the perturbation; and changes in protein stability are all important determinants of the mapping between mRNA and protein expression.     

Mammalian meiosis involves DNA double-strand breaks with 3′ overhangs

Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3′ single-strand overhangs that are active in homology search, repair and formation of recombinant molecules. Are 3′ overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme. We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules. Signals showing 3′ overhangs were observed during wild-type mouse spermatogenesis, but not in Spo11 ^?/? males, which lack meiotic DSBs. In Atm ^?/? mice, abundant positively stained spermatocytes were present, indicating an accumulation of non-repaired DSBs, suggesting the involvement of ATM in repair of meiotic DSBs. Thus the processing of DSBs into 3′ overhangs is common to meiotic cells in mammals and yeast, and probably in all eukaryotes.