Publications by authors named "Javier Cabello-Villegas"

11 Publications

  • Page 1 of 1

Probing RNA structure and interaction dynamics at the single molecule level.

Methods 2019 06 3;162-163:3-11. Epub 2019 Apr 3.

Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

RNA structures and their dynamic fluctuations lie at the heart of understanding key biological process such as transcription, splicing, translation and RNA decay. While conventional bulk assays have proven to identify and characterize key pathway intermediates, the generally dynamic nature of RNA structures renders the information obtained from time and ensemble averaging techniques necessarily lacking in critical details. Here we detail Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS), a method that readily monitors structural fluctuations of single RNA molecules through the repetitive interaction of fluorescent probes with an unlabeled, surface-immobilized RNA target of virtually any length and in any biological context. In addition, we demonstrate the broad applicability of SiM-KARTS by kinetically fingerprinting the binding of cognate tRNA ligand to single immobilized T-box riboswitch molecules. SiM-KARTS represents a valuable tool for probing biologically relevant structure and interaction features of potentially many diverse RNA metabolic pathways.
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http://dx.doi.org/10.1016/j.ymeth.2019.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944401PMC
June 2019

Hierarchical mechanism of amino acid sensing by the T-box riboswitch.

Nat Commun 2018 05 14;9(1):1896. Epub 2018 May 14.

Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.

In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5' half of the T-box riboswitch, but little is known about the mechanism empowering transcriptional control by a small, distal aminoacyl modification. Using single-molecule fluorescence microscopy, we have probed the kinetic and structural underpinnings of tRNA binding to a glycyl T-box riboswitch. We observe a two-step mechanism where fast, dynamic recruitment of tRNA by Stem-I is followed by ultra-stable anchoring by the downstream antiterminator, but only without aminoacylation. Our results support a hierarchical sensing mechanism wherein dynamic global binding of the tRNA body is followed by localized readout of its aminoacylation status by snap-lock-based trapping.
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http://dx.doi.org/10.1038/s41467-018-04305-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951919PMC
May 2018

Role of benzyl alcohol in the unfolding and aggregation of interferon α-2a.

J Pharm Sci 2015 Feb 6;104(2):407-15. Epub 2014 Aug 6.

Department of Pharmaceutical Sciences & Center for Pharmaceutical Biotechnology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, 80045.

Benzyl alcohol (BA) is the most widely used antimicrobial preservative in multidose protein formulations, and has been shown to cause protein aggregation. Our previous work on a model protein cytochrome c demonstrated that this phenomenon occurs via partial unfolding. Here, we examine the validity of these results by investigating the effect of BA on a pharmaceutically relevant protein, interferon α-2a (IFNA2). IFNA2 therapeutic formulations available on the pharmaceutical market contain BA as a preservative. Isothermal aggregation kinetics and temperature scanning demonstrated that BA induced IFNA2 aggregation in a concentration-dependent manner. With increasing concentration of BA, the apparent aggregation temperature of IFNA2 linearly decreased. Denaturant melts measured using protein intrinsic fluorescence and that of the 1-anilinonaphthalene-8-sulfonic acid dye indicated that IFNA2 stability decreased with increasing BA concentration, populating a partially unfolded intermediate. Changes in nuclear magnetic resonance chemical shifts and hydrogen exchange rates identified the structural nature of this intermediate, which correlated with an aggregation "hot-spot" predicted by computational methods. These results indicate that BA induces IFNA2 aggregation by partial unfolding rather than global unfolding of the entire protein, and is consistent with our earlier conclusions from model protein studies.
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http://dx.doi.org/10.1002/jps.24105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312256PMC
February 2015

Effect of antimicrobial preservatives on partial protein unfolding and aggregation.

J Pharm Sci 2013 Feb 20;102(2):365-76. Epub 2012 Nov 20.

Department of Pharmaceutical Sciences & Center for Pharmaceutical Biotechnology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, USA.

One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation 'hot-spot' reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol. All these enhanced the partial protein unfolding that includes a local region which was predicted to be the aggregation 'hot-spot'. The extent of destabilization correlated with the extent of aggregation. Further, we show that stabilizing the 'hot-spot' reduces aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas chlorobutanol causes the least protein aggregation. The same protein region acts as the 'hot-spot' for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others.
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http://dx.doi.org/10.1002/jps.23362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3990441PMC
February 2013

Missense mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-beta aggregates.

Proc Natl Acad Sci U S A 2010 Aug 9;107(34):15069-74. Epub 2010 Aug 9.

Department of Pharmaceutical Sciences, Center for Pharmaceutical Biotechnology, School of Pharmacy, Aurora, CO 80045, USA.

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations--L54R, A168D, A171P, and Y231N--on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded alpha-helical protein in solution, as is evident from its alpha-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR (15)N-(1)H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal (T(1)) and transverse (T(2)) relaxation experiments. Compared to WT, three mutants--L54R, A168D, and A171P--show a decreased alpha-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a "molten globule" state. In contrast, Y231N has an alpha-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-beta structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.
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http://dx.doi.org/10.1073/pnas.1008818107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930578PMC
August 2010

Role of partial protein unfolding in alcohol-induced protein aggregation.

Proteins 2010 Sep;78(12):2625-37

Department of Pharmaceutical Sciences and Center for Pharmaceutical Biotechnology, School of Pharmacy, University of Colorado Denver, 12700 E 19th Avenue, C238-P15, Aurora, Colorado 80045, USA.

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as nondisease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins.
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http://dx.doi.org/10.1002/prot.22778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927387PMC
September 2010

Solution structure of psi32-modified anticodon stem-loop of Escherichia coli tRNAPhe.

Nucleic Acids Res 2005 23;33(22):6961-71. Epub 2005 Dec 23.

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA.

Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem-loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg2+ and dimethylallyl modification of A37 N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, psi32, psi39, and A37 C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of psi32 on the anticodon stem-loop from E.coli tRNA(Phe). The psi32 modification does not significantly alter the structure of the anticodon stem-loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs psi32-A38 and U33-A37 and the base of psi32 stacks between U33 and A31. The glycosidic bond of psi32 is in the anti configuration and is paired with A38 in a Watson-Crick geometry, unlike residue 32 in most crystal structures of tRNA. The psi32 modification increases the melting temperature of the stem by approximately 3.5 degrees C, although the psi32 and U33 imino resonances are exchange broadened. The results suggest that psi32 functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.
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http://dx.doi.org/10.1093/nar/gki1004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1322268PMC
January 2006

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor.

RNA 2004 Sep;10(9):1388-98

Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, CCR, National Cancer Institute-Frederick, National Institutes of Health, MD 21702, USA.

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.
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http://dx.doi.org/10.1261/rna.7020804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1370626PMC
September 2004

Periodicity in residual dipolar couplings and nucleic acid structures.

J Am Chem Soc 2004 Feb;126(7):1938-9

Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.

The periodicity in nucleic acid duplex structures is shown to be correlated to the periodicity in residual dipolar couplings (RDCs) in the form of an "RDC wave". This "RDC wave" is characteristic of the alignment of the duplex in the magnetic field, and hence fitting of the data allows the duplex global orientation (, Phi) to be extracted. Further, because the "RDC wave" is fit as a data set of a corresponding secondary structure element, the degeneracy problem is greatly reduced. Consequently, with the global orientation (, Phi) determined, local bond vector conformations are defined. The fit is demonstrated in the examples of the imino RDCs of the negative regulator of splicing RNA fragment (NRS23) and for the C1'H1' RDCs of the Dickerson dodecamer.
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http://dx.doi.org/10.1021/ja039446gDOI Listing
February 2004

Metal ion stabilization of the U-turn of the A37 N6-dimethylallyl-modified anticodon stem-loop of Escherichia coli tRNAPhe.

Biochemistry 2004 Jan;43(1):55-66

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA.

Nucleoside base modifications can alter the structures, dynamics, and metal ion binding properties of transfer RNA molecules and are important for accurate aminoacylation and for maintaining translational fidelity and efficiency. The unmodified anticodon stem-loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg(2+) and dimethylallyl modification of A(37) N6 disrupt the loop conformation and increase the mobility of the loop and loop-proximal nucleotides. We have used NMR spectroscopy to investigate the binding and structural effects of multivalent cations on the unmodified and dimethylallyl-modified anticodon stem-loops from E. coli tRNA(Phe). The divalent cation binding sites were probed using Mn(2+) and Co(NH(3))(6)(3+). These ions bind along the major groove of the stem and associate with the anticodon loop on the major groove side in a nonspecific manner. Co(NH(3))(6)(3+) stabilizes the U-turn conformation of the loop in the dimethylallyl-modified molecule, and the chemical shift changes that accompany Co(NH(3))(6)(3+) binding are similar to those observed with the addition of Mg(2+). The base-phosphate and base-2'-OH hydrogen bonds that characterize the UNR U-turn motif lead to spectral signatures in the form of unusual (15)N and (1)H chemical shifts and reduced solvent exchange of the U(33) 2'-OH and N3H protons. The unmodified molecule also displays spectral features of the U-turn fold in the presence of Co(NH(3))(6)(3+), but the loop has additional conformations and is dynamic. The results indicate that charge neutralization by a polyvalent cation is sufficient to promote formation of the U-turn fold. However, base modification is necessary to destabilize competing alternative conformers even for a purine-rich loop sequence that is predicted to have strongly favorable base stacking energy.
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http://dx.doi.org/10.1021/bi0353676DOI Listing
January 2004

Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe).

J Mol Biol 2002 Jun;319(5):1015-34

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA.

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.
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http://dx.doi.org/10.1016/S0022-2836(02)00382-0DOI Listing
June 2002
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