Ricardo Bernal - Academia.edu (original) (raw)

Papers by Ricardo Bernal

New Journal of Chemistry, 2018

Sodium rhodizonate mediated green synthesis of gold, silver, platinum, and palladium nanoparticle... more Sodium rhodizonate mediated green synthesis of gold, silver, platinum, and palladium nanoparticles and their catalytic reduction of 4-nitrophenol and methyl orange.

Frontiers in molecular biosciences, 2018

Chaperonins are macromolecular complexes found throughout all kingdoms of life that assist unfold... more Chaperonins are macromolecular complexes found throughout all kingdoms of life that assist unfolded proteins reach a biologically active state. Historically, chaperonins have been classified into two groups based on sequence, subunit structure, and the requirement for a co-chaperonin. Here, we present a brief review of chaperonins that can form double- and single-ring conformational intermediates in their protein-folding catalytic pathway. To date, the bacteriophage encoded chaperonins ϕ-EL and OBP, human mitochondrial chaperonin and most recently, the bacterial groEL/ES systems, have been reported to form single-ring intermediates as part of their normal protein-folding activity. These double-ring chaperonins separate into single-ring intermediates that have the ability to independently fold a protein. We discuss the structural and functional features along with the biological relevance of single-ring intermediates in cellular protein folding. Of special interest are the ϕ-EL and O...

ACS applied materials & interfaces, Jan 20, 2017

Remarkable recent advances on Au25(SR)18 nanoclusters have led to significant applications in cat... more Remarkable recent advances on Au25(SR)18 nanoclusters have led to significant applications in catalysis, sensing, and magnetism. However, the existing synthetic routes are complicated, particularly for the water-soluble Au25(SG)18 nanoclusters. Here, we report a single-step concentration and temperature-controlled method for rapid synthesis of the Au25(SG)18 nanoclusters in as little as 2 h without the need for low-temperature reaction or even stirring. A systematic time-based investigation was carried out to study the effects of volume, concentration, and temperature on the synthesis of these nanoclusters. Further, we discovered for the first time that the Au25(SG)18 nanoclusters exhibit excellent photothermal activities in achieving 100% cell death for MDA-MB-231 breast cancer cells at a power of 10 W/cm2 using an 808 nm laser source, demonstrating applications toward photothermal therapy.

Cell cycle (Georgetown, Tex.), Jan 8, 2017

The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding ... more The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 Å resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a non-native substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used ...

Structure, 2016

Highlights d The 4EL chaperonin is the first of only two groEL orthologs encoded by a phage d ATP... more Highlights d The 4EL chaperonin is the first of only two groEL orthologs encoded by a phage d ATP hydrolysis induces 4EL chaperonin to dissociate into two closed single rings d The 4EL chaperonin can fold substrate proteins without a cochaperonin d The 4EL chaperonin characteristics resemble both group I and II chaperonins

Structure (London, England : 1993), Jan 28, 2016

Chaperonins are essential biological complexes assisting protein folding in all kingdoms of life.... more Chaperonins are essential biological complexes assisting protein folding in all kingdoms of life. Whereas homooligomeric bacterial GroEL binds hydrophobic substrates non-specifically, the heterooligomeric eukaryotic CCT binds specifically to distinct classes of substrates. Sulfolobales, which survive in a wide range of temperatures, have evolved three different chaperonin subunits (α, β, γ) that form three distinct complexes tailored for different substrate classes at cold, normal, and elevated temperatures. The larger octadecameric β complexes cater for substrates under heat stress, whereas smaller hexadecameric αβ complexes prevail under normal conditions. The cold-shock complex contains all three subunits, consistent with greater substrate specificity. Structural analysis using crystallography and electron microscopy reveals the geometry of these complexes and shows a novel arrangement of the α and β subunits in the hexadecamer enabling incorporation of the γ subunit.

Molecular and Cellular Biology, 1996

NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in ... more NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK...

Oncotarget

The regulation of steroidogenic hormone receptor-mediated activity plays an important role in the... more The regulation of steroidogenic hormone receptor-mediated activity plays an important role in the development of hormone-dependent cancers. For example, during prostate carcinogenesis, the regulatory function played by the androgen receptor is often converted from a growth suppressor to an oncogene thus promoting prostate cancer cell survival and eventual metastasis. Within the cytoplasm, steroid hormone receptor activity is regulated by the Hsp90 chaperone in conjunction with a series of co-chaperone proteins. Collectively, Hsp90 and its binding associates form a large heteromeric complex that scaffold the fully mature receptor for binding with the respective hormone. To date our understanding of the interactions between Hsp90 with the various TPR domain-containing co-chaperone proteins is limited due to a lack of available structural information. Here we present the stable formation of Hsp90(2)-FKBP52(1)- HOP(2) and Hsp90(2)-FKBP52(1)-p23(2)-HOP(2) complexes as detected by immunop...

Bacteriophage, 2013

The bacteriophage EL is a virus that specifically attacks the human pathogen Pseudomonas aerugino... more The bacteriophage EL is a virus that specifically attacks the human pathogen Pseudomonas aeruginosa. This phage carries a large genome that encodes for its own chaperonin which presumably facilitates the proper folding of phage proteins independently of the host chaperonin system. EL also encodes a lysin enzyme, a critical component of the lytic cycle that is responsible for digesting the peptidoglycan layer of the host cell wall. Previously, this lysin was believed to be a substrate of the chaperonin encoded by phage EL. In order to characterize the activity of the EL lysin, and to determine whether lysin activity is contingent on chaperonin-mediated folding, a series of peptidoglycan hydrolysis activity assays were performed. Results indicate that the EL-encoded lysin has similar enzymatic activity to that of the Gallus gallus lysozyme and that the EL lysin folds into a functional enzyme in the absence of phage chaperonin and should not be considered a substrate.

The EMBO Journal, 2005

The crystal structure of subunit F of vacuole-type ATPase/ synthase (prokaryotic V-ATPase) was de... more The crystal structure of subunit F of vacuole-type ATPase/ synthase (prokaryotic V-ATPase) was determined to of 2.2 Å resolution. The subunit reveals unexpected structural similarity to the response regulator proteins that include the Escherichia coli chemotaxis response regulator CheY. The structure was successfully placed into the low-resolution EM structure of the prokaryotic holo-V-ATPase at a location indicated by the results of crosslinking experiments. The crystal structure, together with the single-molecule analysis using fluorescence resonance energy transfer, showed that the subunit F exhibits two conformations, a 'retracted' form in the absence and an 'extended' form in the presence of ATP. Our results postulated that the subunit F is a regulatory subunit in the V-ATPase.

Structure, 2004

sequence identity (Table 1) (Gruber et al., 2001; Wilms et al., 1996). Eukaryotic V-ATPases have ... more sequence identity (Table 1) (Gruber et al., 2001; Wilms et al., 1996). Eukaryotic V-ATPases have further evolved additional subunit components that increase their structural and apparent regulatory complexity in comparison to the A-ATPases (Forgac, 1999). The most significant

Science, 2011

The effect of lipids and nucleotides on the soluble head domain and membrane base domain is exami... more The effect of lipids and nucleotides on the soluble head domain and membrane base domain is examined in an intact adenosine triphosphatase.

PLoS ONE, 2010

Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical grad... more Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical gradients and preserving pH-dependent cellular compartments by way of proton translocation across the membrane. V-ATPases employ a dynamic rotary mechanism that is driven by ATP hydrolysis and the central rotor stalk. Regulation of this rotational catalysis is the result of a reversible V 1 V o-domain dissociation that is required to preserve ATP during instances of cellular starvation. Recently the method by which the free V 1-ATPase abrogates the hydrolytic breakdown of ATP upon dissociating from the membrane has become increasingly clear. In this instance the central stalk subunit F adopts an extended conformation to engage in a bridging interaction tethering the rotor and stator components together. However, the architecture by which this mechanism is stabilized has remained ambiguous despite previous work. In an effort to elucidate the method by which the rotational catalysis is maintained, the architecture of the peripheral stalks and their respective binding interactions was investigated using cryo-electron microscopy. In addition to confirming the bridging interaction exuded by subunit F for the first time in a eukaryotic V-ATPase, subunits C and H are seen interacting with one another in a tight interaction that provides a base for the three EG peripheral stalks. The formation of a CE 3 G 3 H sub-assembly appears to be unique to the dissociated V-ATPase and highlights the stator architecture in addition to revealing a possible intermediate in the assembly mechanism of the free V 1-ATPase.

Nature Structural & Molecular Biology, 2010

Proton-translocating ATPases are ubiquitous protein complexes that couple ATP catalysis with prot... more Proton-translocating ATPases are ubiquitous protein complexes that couple ATP catalysis with proton translocation via a rotary catalytic mechanism. The peripheral stalks are essential components that counteract torque generated from proton translocation during ATP synthesis or from ATP hydrolysis during proton pumping. Despite their essential role, the peripheral stalks are the least conserved component of the complexes, differing substantially between subtypes in composition and stoichiometry. We have determined the crystal structure of the peripheral stalk of the A-type ATPase/synthase from Thermus thermophilus consisting of subunits E and G. The structure contains a heterodimeric right-handed coiled coil, a protein fold never observed before. We have fitted this structure into the 23-Å resolution electron microscopy density of the intact A-ATPase complex, revealing the precise location of the peripheral stalk and new implications for the function and assembly of proton-translocating ATPases. Proton-translocating ATPases (H +-ATPases) are rotary enzymes that couple proton (or Na +) translocation across membranes with ATP synthesis or hydrolysis 1-4. There are three evolutionarily related subtypes of these protein complexes that are categorized as F-, V-and A-type ATPases on the basis of their function and taxonomic origin. F-type ATPases, better known as F 1 F o ATP synthases, use energy from proton translocation across an electrochemical gradient to synthesize ATP 3. In contrast, vacuolar or V-type ATPases work in reverse by actively pumping protons through membranes using energy derived from ATP Correspondence should be addressed to D.S.

Journal of Structural Biology, 2001

An algorithm has been developed for placing three-dimensional atomic structures into appropriatel... more An algorithm has been developed for placing three-dimensional atomic structures into appropriately scaled cryoelectron microscopy maps. The first stage in this process is to conduct a threedimensional angular search in which the center of gravity of an X-ray crystallographically determined structure is placed on a selected position in the cryoelectron microscopy map. The quality of the fit is measured by the sum of the density at each atomic position. The second stage is to refine the three angles and three translational parameters for the best (usually 25 to 100) fits. Useful criteria for this refinement include the sum of densities at atomic sites, the lack of atoms in negative or low density, the absence of atomic clashes between symmetry-related positions of the atomic structure, and the distances between identifiable features in the map and their positions on the fitted atomic structure. These refinements generally lead to a convergence of the originally chosen, top scoring fits to just a few (about 3 to 8) acceptable possibilities. Usually, the best remaining fit is clearly superior to any of the others.

Journal of Molecular Biology, 2003

Bacteriophage a3 is a member of the Microviridae, a family of small, singlestranded, icosahedral ... more Bacteriophage a3 is a member of the Microviridae, a family of small, singlestranded, icosahedral phages that include fX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108 S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The a3 "open" procapsid structural intermediate was determined to 15 Å resolution by cryo-electron microscopy (cryo-EM). Unlike the fX174 "closed" procapsid and the infectious virion, the a3 open procapsid has 30 Å wide pores at the 3-fold vertices and 20 Å wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30 Å wide pores. The structure of the a3 mature virion was solved to 3.5 Å resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the a3 and fX174 virion structures are in the spike and the DNA-binding proteins. The a3 pentameric spikes have a rotation of 3.58 compared to those of fX174. The a3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the fX174 J protein, retains its carboxyterminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in a3 compared to fX174, allowing the building of about 10% of the ribose-phosphate backbone.

Journal of Molecular Biology, 2004

Packaging of viral genomes into their respective capsids requires partial neutralization of the h... more Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages fX174, G4, and a3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The fX174 DNA-binding protein, J, is 13 amino acid residues longer than the a3 and G4 J proteins by virtue of an additional nucleic acidbinding domain at the amino terminus. Chimeric fX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric a3 and G4 phages, containing the fX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of a3 packaged with fX174 J protein was determined to 3.5 Å resolution and compared with the previously determined structures of fX174 and a3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type a3 virion proteins. The amino-terminal region of the fX174 J protein, which is missing from wild-type a3 virions, is mostly disordered in the a3 chimera. The differences observed between solution properties of wild-type fX174, wild-type a3, and a3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the fX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection. Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection.

Journal of Molecular Biology, 1999

An empty precursor particle called the procapsid is formed during assembly of the single-stranded... more An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage fX174. Assembly of the fX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of à`c losed'' procapsid particle has been determined to 3.5 A Ê resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A Ê resolution electron microscopic reconstruction of thè`o pen'' procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly a-helical fold and displays remarkable conformational variability. We report here an improved and re®ned structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.

Journal of Chromatography B, 2010

Hsp90 is a ubiquitous molecular chaperone that plays a key role in the malignant development of h... more Hsp90 is a ubiquitous molecular chaperone that plays a key role in the malignant development of hormone-dependent pathologies such as cancer. An important role for Hsp90 is to facilitate the stable binding of steroid hormones to their respective receptors enabling the ligand-based signal to be carried to the nucleus and ultimately resulting in the up-regulation of gene expression. Along with Hsp90, this dynamic and transient process also involves the recruitment of additional proteins and co-chaperones that add further stability to the mature receptor-chaperone complex. In the work presented here, we describe four new protocols for the bacterial over-expression and column chromatographic purification of the human p23, FKBP52, HOP and SGT␣ proteins. Each of these proteins plays a distinct role in the steroid hormone receptor regulatory cycle. Affinity, ion-exchange and size-exclusion techniques were used to produce target yields greater than 50 mg/L of cultured media, with each purified sample reaching near absolute sample homogeneity. These results reveal a reliable system for the production of p23, FKBP52, HOP and SGT␣ substrate proteins for use in the investigation of the Hsp90-associated protein interactions of the steroid hormone receptor cycle.