Carlos Amero

Fast Two-Dimensional NMR Spectroscopy of High Molecular Weight Protein Assemblies

An optimized NMR experiment that combines the advantages of methyl-TROSY and SOFAST-HMQC has been developed. It allows the recording of high quality methyl (1)H-(13)C correlation spectra of protein assemblies of several hundreds of kDa in a few seconds. The SOFAST-methyl-TROSY-based experiment offers completely new opportunities for the study of structural and dynamic changes occurring in molecular nanomachines while they perform their biological function in vitro.

A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies

Obtaining sequence-specific assignments remains a major bottleneck in solution NMR investigations of supramolecular structure, dynamics and interactions. Here we demonstrate that resonance assignment of methyl probes in high molecular weight protein assemblies can be efficiently achieved by combining fast NMR experiments, residue-type-specific isotope-labeling and automated site-directed mutagenesis. The utility of this general and straightforward strategy is demonstrated through the characterization of intermolecular interactions involving a 468-kDa multimeric aminopeptidase, PhTET2.

Structural basis for the inhibition of truncated islet amyloid polypeptide aggregation by Cu(II): insights into the bioinorganic chemistry of type II diabetes.

Type 2 diabetes (T2D) is one of the most common chronic diseases, affecting over 300 million people worldwide. One of the hallmarks of T2D is the presence of amyloid deposits of human islet amyloid polypeptide (IAPP) in the islets of Langerhans of pancreatic β-cells. Recent reports indicate that Cu(II) can inhibit the aggregation of human IAPP, although the mechanism for this inhibitory effect is not clear. In this study, different spectroscopic techniques and model fragments of IAPP were employed to shed light on the structural basis for the interaction of Cu(II) with human IAPP. Our results show that Cu(II) anchors to His18 and the subsequent amide groups toward the C-terminal, forming a complex with an equatorial coordination mode 3N1O at physiological pH. Cu(II) binding to truncated IAPP at the His18 region is the key event for its inhibitory effect in amyloid aggregation. Electron paramagnetic resonance studies indicate that the monomeric Cu(II)-IAPP(15-22) complex differs significantly from Cu(II) bound to mature IAPP(15-22) fibers, suggesting that copper binding to monomeric IAPP(15-22) competes with the conformation changes needed to form β-sheet structures, thus delaying fibril formation. A general mechanism is proposed for the inhibitory effect of copper and other imidazole-binding metal ions in IAPP amyloid formation, providing further insights into the bioinorganic chemistry of T2D.

A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties

Background: Late embryogenesis-abundant proteins accumulate under water-deficit and are widely distributed in plants and anhydrobiotic organisms. Results: A common bean group-6 LEA protein shows structural disorder, adopts helicity under water deficit or high molecular crowding, and presents oligomeric forms. Conclusion: PvLEA6 protein adopts different conformations and/or a quaternary structure depending on its environment. Significance: Similar characteristics might be present in different LEA proteins, which could be relevant to their function. Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.

Copper and Zinc Ions Specifically Promote Nonamyloid Aggregation of the Highly Stable Human γ-D Crystallin.

Cataract is the leading cause of blindness in the world. It results from aggregation of eye lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. Copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy lens, and a variety of experimental and epidemiological studies implicate metals as potential etiological agents for cataract. The natively monomeric, β-sheet rich human γD (HγD) crystallin is one of the more abundant proteins in the core of the lens. It is also one of the most thermodynamically stable proteins in the human body. Surprisingly, we found that both Cu(II) and Zn(II) ions induced rapid, nonamyloid aggregation of HγD, forming high-molecular-weight light-scattering aggregates. Unlike Zn(II), Cu(II) also substantially decreased the thermal stability of HγD and promoted the formation of disulfide-bridged dimers, suggesting distinct aggregation mechanisms. In both cases, however, metal-induced aggregation depended strongly on temperature and was suppressed by the human lens chaperone αB-crystallin (HαB), implicating partially folded intermediates in the aggregation process. Consistently, distinct site-specific interactions of Cu(II) and Zn(II) ions with the protein and conformational changes in specific hinge regions were identified by nuclear magnetic resonance. This study provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease.

Inhibition of Light Chain 6aJL2-R24G Amyloid Fiber Formation Associated with Light Chain Amyloidosis

Light chain amyloidosis (AL) is a deadly disease characterized by the deposition of monoclonal immunoglobulin light chains as insoluble amyloid fibrils in different organs and tissues. Germ line λ VI has been closely related to this condition; moreover, the R24G mutation is present in 25% of the proteins of this germ line in AL patients. In this work, five small molecules were tested as inhibitors of the formation of amyloid fibrils from the 6aJL2-R24G protein. We have found by thioflavin T fluorescence and transmission electron microscopy that EGCG inhibits 6aJL2-R24G fibrillogenesis. Furthermore, using nuclear magnetic resonance spectroscopy, dynamic light scattering, and isothermal titration calorimetry, we have determined that the inhibition is due to binding to the protein in its native state, interacting mainly with aromatic residues.

Ligand-Induced Changes in the Structure and Dynamics of Escherichia coli Peptide Deformylase

Peptide deformylase (PDF) is an enzyme that is responsible for removing the formyl group from nascently synthesized polypeptides in bacteria, attracting much attention as a potential target for novel antibacterial agents. Efforts to develop potent inhibitors of the enzyme have progressed on the basis of classical medicinal chemistry, combinatorial chemistry, and structural approaches, yet the validity of PDF as an antibacterial target hangs, in part, on the ability of inhibitors to selectively target this enzyme in favor of structurally related metallohydrolases. We have used (15)N NMR spectroscopy and isothermal titration calorimetry to investigate the high-affinity interaction of EcPDF with actinonin, a naturally occurring potent EcPDF inhibitor. Backbone amide chemical shifts, residual dipolar couplings, hydrogen-deuterium exchange, and (15)N relaxation reveal structural and dynamic effects of ligand binding in the immediate vicinity of the ligand-binding site as well as at remote sites. A comparison of the crystal structures of free and actinonin-bound EcPDF with the solution data suggests that most of the consequences of the ligand binding to the protein are lost or obscured during crystallization. The results of these studies improve our understanding of the thermodynamic global minimum and have important implications for structure-based drug design.

Moonlighting Peptides with Emerging Function

Hunter-killer peptides combine two activities in a single polypeptide that work in an independent fashion like many other multi-functional, multi-domain proteins. We hypothesize that emergent functions may result from the combination of two or more activities in a single protein domain and that could be a mechanism selected in nature to form moonlighting proteins. We designed moonlighting peptides using the two mechanisms proposed to be involved in the evolution of such molecules (i.e., to mutate non-functional residues and the use of natively unfolded peptides). We observed that our moonlighting peptides exhibited two activities that together rendered a new function that induces cell death in yeast. Thus, we propose that moonlighting in proteins promotes emergent properties providing a further level of complexity in living organisms so far unappreciated.

Information flow and protein dynamics: the interplay between nuclear magnetic resonance spectroscopy and molecular dynamics simulations

Proteins participate in information pathways in cells, both as links in the chain of signals, and as the ultimate effectors. Upon ligand binding, proteins undergo conformation and motion changes, which can be sensed by the following link in the chain of information. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations represent powerful tools for examining the time-dependent function of biological molecules. The recent advances in NMR and the availability of faster computers have opened the door to more detailed analyses of structure, dynamics, and interactions. Here we briefly describe the recent applications that allow NMR spectroscopy and MD simulations to offer unique insight into the basic motions that underlie information transfer within and between cells.

Solution structure of 6aJL2 and 6aJL2-R24G amyloidogenics light chain proteins.

AL amyloidosis is the most common amyloid systemic disease and it is characterized by the deposition of immunoglobulin light chain amyloid fibers in different organs, causing organ failure. The immunoglobulin light chain germinal line 6a has been observed to over-express in AL patients, moreover, it was observed that, out of these amyloidogenic proteins, 25% present a mutation of an Arg to Gly in position 24. In vitro studies have shown that this mutation produces proteins with a higher amyloid fiber propensity. It was proposed that this difference was due, in part, to the formation of a non-canonical structural element. In order to get a more detailed understanding of the structural and dynamic properties that govern the amyloid fibers formation process, we have determined the solution structure by NMR for the two constructs, showing that the difference in amyloid fibril formation is not due to sequence or structure.