A5000136040- Nanopore and Nanochannel Transport Studies
- Protein Structure and Dynamics
- Enzyme Structure and Function
- Force Microscopy Techniques and Applications
- Lipid Membrane Structure and Behavior
- RNA and protein synthesis mechanisms
- Microfluidic and Capillary Electrophoresis Applications
- Redox biology and oxidative stress
- Mass Spectrometry Techniques and Applications
- Ubiquitin and proteasome pathways
- Bacterial Genetics and Biotechnology
- Microfluidic and Bio-sensing Technologies
- Monoclonal and Polyclonal Antibodies Research
- Heat shock proteins research
- 14-3-3 protein interactions
- Ion-surface interactions and analysis
- Erythrocyte Function and Pathophysiology
- Bacteriophages and microbial interactions
- Ion channel regulation and function
- Biomedical Research and Pathophysiology
- RNA Interference and Gene Delivery
- thermodynamics and calorimetric analyses
- ATP Synthase and ATPases Research
- Protein Interaction Studies and Fluorescence Analysis
- Membrane-based Ion Separation Techniques
Instituto Biofisika
2017-2023
Fundación Biofísica Bizkaia
2020-2021
University of Oxford
2012-2014
Universidad de Granada
2006-2012
Porins are β-barrel outer-membrane proteins through which small solutes and metabolites diffuse that also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) assembles a cytotoxic translocon at surface of Escherichia coli incorporates trimeric porin OmpF. Formation involved ColE9's unstructured N-terminal domain threading in opposite directions two OmpF subunits, capturing its target TolB on other side membrane fixed orientation triggers import. Thus, an...
Abstract Protein unfolding and translocation through pores occurs during trafficking between organelles, protein degradation bacterial toxin delivery. In vivo , co-translocational can be affected by the end of polypeptide that is threaded into pore first. Recently, we have shown followed in a model system at single-molecule level, thereby unravelling molecular steps their kinetics. Here, show kinetics substrate thioredoxin, when pulled an α-haemolysin pore, differ markedly depending on...
Human triosephosphate isomerase deficiency is a rare autosomal disease that causes premature death of homozygous individuals. The most frequent mutation leads to this illness in position 104, which involves conservative change Glu for Asp. Despite the extensive work has been carried out on E104D mutant enzyme hemolysates and whole cells, molecular basis poorly understood. Here, we show purified, recombinant E104D, while exhibiting normal catalytic activity, shows impairments formation active...
Protein interactions with specific DNA sequences are crucial in the control of gene expression and regulation replication. Single-molecule methods offer excellent capabilities to unravel mechanism kinetics these interactions. Here, we develop a nanopore approach where target sequence is contained hairpin followed by ssDNA. This system allows DNA–protein complexes be distinguished from bare molecules as they pulled through single detector, providing both equilibrium kinetic information. We...
Tyrosine hydroxylase (TH), the rate-limiting enzyme in synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain TH also involved interaction with lipid membranes. We investigated its different partners, both unphosphorylated (TH-(1–43)) and Ser19-phosphorylated (THp-(1–43)) states surface plasmon resonance. THp-(1–43) showed high affinity for proteins (Kd ∼ 0.5 μm 14-3-3γ -ζ 7 14-3-3η). domains bind negatively charged membranes...
Abstract It is widely recognized that enhancement of protein stability an important biotechnological goal. However, some applications at least, could actually benefit from being strongly dependent on a suitable environment variable, in such way enhanced or decreased be realized as required. In therapeutic applications, for instance, long shelf‐life under storage conditions may convenient, but sufficiently fast degradation the after it has performed planned molecular task vivo avoid side...
A technology capable of sequencing individual protein molecules would revolutionize our understanding biological processes. Nanopore can analyze single heteropolymer such as DNA by measuring the ionic current flowing through a nanometer hole made in an electrically insulating membrane. This is sensitive to monomer sequence. However, proteins are remarkably complex and identifying residue change remains challenge. In this work, I show that simple neural networks be trained recognize mutants....
Abstract Bacterial conjugation is the main mechanism for dissemination of antibiotic resistance genes. A single DNA strand conjugative plasmid transferred across bacterial membranes covalently bound to a large multi-domain protein, named relaxase, which must be unfolded traverse secretion channel. Two tyrosine residues relaxase (Y18 and Y26 in TrwC) play an important role processing DNA. We have used nanopore technology uncover unfolding states that take place during translocation...
Recent work has shown that proteins can tolerate hydrophobic-to-ionizable-residue mutations. Here, we provide experimental evidence the essential properties (pK value, protonation state, local dynamics) of buried ionizable groups in be efficiently modulated through rational design surface charge distribution, thus paving way for protein engineering exploitation burial.
Abstract Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however dynamics proteins during remain largely obscure. Here we study, single-molecule level, folding landscape a model while forced to translocate transmembrane pore. We use DNA tag drive α-hemolysin pore under quantifiable force produced by an applied electric potential. Using voltage-quench approach find that...
Protein physicochemical properties must undergo complex changes during evolution, as a response to modifications in the organism environment, result of proteins taking up new roles or because need cope with evolution molecular interacting partners. Recent work has emphasized role stability and stability–function trade-offs these protein adaptation processes. In present study, on other hand, we report that combinations few conservative, high-frequency-of-fixation mutations thioredoxin...
A large collection of structural snapshots along a full catalytic cycle Escherichia coli thioredoxin reductase (TrxR) has been generated and characterized using combination theoretical methods. Molecular models were built starting from the available X-ray crystallographic structures dimeric wild-type TrxR in flavin-oxidizing conformation C135S mutant enzyme flavin-reducing "trapped" by cross-link between Cys138 Cys32 C35S (Trx). The transition these two extreme states, which is shown to be...
Abstract Understanding protein folding under conditions similar to those found in vivo remains challenging. Folding occurs mainly vectorially as a polypeptide emerges from the ribosome or membrane translocon. Protein during translocation is particularly difficult study. Here, we describe single-molecule method characterize folded state of individual proteins after translocation, by monitoring ionic current passing through pore. We tag both N and C termini model protein, thioredoxin, with...
Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of substantiate importance investigating molecular mechanisms membrane interaction. By applying surface plasmon resonance we here show binding phospholipid bilayers is stimulated when 14-3-3γ complexed with its...