The two nitrile groups at the wings of the nonnucleoside HIV-1 reverse transcriptase (RT) inhibitor TMC278 are both identified in high-sensitivity 2D IR spectroscopy experiments of the HIV-1 RT/ TMC278 complex. The vibrational spectra indicate that the two arms of the inhibitor sense quite different environments within the hydrophobic pocket. The vibrational relaxation of the two arms are almost equal at 3 ps from model studies. The 2D IR spectra expose a significant distribution of nitrile frequencies that diffuse at equilibrium on ultrafast time scales ranging from hundreds of femtoseconds to tens of picoseconds. The slow spectral diffusion of the cyanovinyl arm of the inhibitor is attributed to its interaction with the backbone and side chains in the hydrophobic tunnel. The results show that the inhibitor cyano modes lose memory of their structural configurations relative to the hydrophobic pocket within tens of picoseconds. The cross-peaks between the two arms of the drug are tentatively attributed to relaxation of the nitrile state with both arms excited.2D IR spectroscopy ͉ ultrafast protein response ͉ vibrational relaxation R everse transcriptase (RT) enables the conversion of a singlestranded RNA into a double-stranded DNA in a process that is essential for viral replication. The nonnucleoside inhibitors of HIV-1 RT (NNRTIs) bind in a hydrophobic pocket between two -sheets, one containing the polymerase active site and the other containing the primer grip, a structural element that is involved in positioning the 3Ј primer terminus for catalysis (1). Resistance to NNRTIs can arise from mutations in the residues in and around the binding pocket (2). An NNRTI that has strategic flexibility can overcome the effects of drug-resistance mutations by adapting its conformation and position to accommodate mutation-induced changes in the binding pocket (3). The diarylpyrimidine (DAPY) NNRTI TMC278 (4) is a flexible molecule and inhibits a broad spectrum of NNRTI-resistant mutants. Questions then arise as to freedom of motion of the inhibitor and the time scales of such structural reorganizations. To obtain answers there is a need for time-sensitive probes of structure within the pocket that can respond to motions of the drug and the nearby residues. One set of probes that do not perturb the structure and that span the short time regime are the vibrations of the chemical bonds of the inhibitor. They sense the fluctuations of the surrounding structure through the time correlation functions of their vibrational frequencies. These correlation functions can be measured with the methods of 2D IR spectroscopy (5), and the present work is an application of 2D IR spectroscopy aimed at exposing dynamical aspects of the enzyme inhibitor complex at the chemical bond-length scale. There have been many recent experimental and theoretical advances showing that these methods can expose novel aspects of ultrafast processes in biological assemblies (6-18). The present article on the vibrational spectra and dynamics of an HIV-1 RT i...
Two dimensional vibrational echo spectroscopy has previously been applied to structural determination of small peptides. Here we extend the technique to a more complex, biologically significant system: the homodimeric transmembrane dimer from the α-chain of the integrin αIIbβ3. We prepared micelle suspensions of the pair of 30-residue chains that span the membrane in the native structure, with varying levels of heavy (13C=18O) isotopes substituted in the backbone of the central 10th through 20th positions. The constraints derived from vibrational coupling of the precisely spaced heavy residues led to determination of an optimized structure from a range of model candidates: Glycine residues at the 12th, 15th and 16th positions form a tertiary contact in parallel right handed helix dimers with crossing angles of −58° ± 9° and interhelical distances of 7.7 ± 0.5 Å. The frequency correlation established the dynamical model used in the analysis and it indicated the absence of mobile water associated with labeled residues. Delocalization of vibrational excitations between the helices was also quantitatively established.
The CN vibrations of two aromatic nitriles, cinnamonitrile, PhCH=CH-CN and benzonitrile, PhCN, representative of components of common enzyme inhibitors, are examined by two dimensional infrared spectroscopy. In methanol, these spectra display cross peaks between the two CN components whose evolution exposes the few picosecond (4.5 ps for CIN and 5.3 ps for BN) equilibrium dynamics of hydrogen bond making and breaking. The main features of the 2D IR spectra are reproduced by simulations only with exchange incorporated. The lowest free energy state is the non-hydrogen bonded form. Both alkyl and aryl nitriles have now shown this picosecond exchange process.
Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.
Conspectus The development of experiments that can generate molecular movies of changing chemical structures is a major challenge for physical chemistry. But to realize this dream, we not only need to significantly improve existing approaches, but we also must invent new technologies .. Most of the known protein structures have been determined by X-ray diffraction and to lesser extent by NMR. Though powerful, X-ray diffraction presents limitations for acquiring time dependent structures. In the case of NMR, ultrafast equilibrium dynamics might be inferred from lineshapes, but the structures of conformations interconverting on such time scales are not realizable. This Account highlights two dimensional infrared spectroscopy (2D IR), in particular the 2D vibrational echo, as an approach to time resolved structure determination. We outline the use of the 2D IR method to completely determine the structure of a protein of the integrin family in a time window of few picoseconds. As a transmembrane protein, this class of structures has proved particularly challenging for the established structural methodologies of x-ray crystallography and NMR. We describe the challenges facing multidimensional spectroscopy and compare it with some other methods of structural biology. Then we succinctly discuss the basic principles of 2D IR methods as they relate to time domain and frequency domain experimental and theoretical properties required for protein structure determination. By means of the example of the transmembrane protein, we describe the essential aspects of combined carbon-13 oxygen-18 isotope labels to create vibrational resonance pairs that allow the determination of protein and peptide structures in motion. Finally, we propose a three dimensional structure of the αIIb transmembrane homodimer that includes optimum locations of all side chains and backbone atoms of the protein. Delocalization among 13C=18O residues on different helices. The vibrational excitation is transferred between modes on different helices on the coherent energy transfer time π/2β.
Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.
Highlights d Rac1 immobilization at the lamellipodium tip correlates with its activation d Rac1 immobilization depends on effector binding, including WRC d RhoA does not display selective immobilization at the lamellipodium tip d Local Rac1 activation at the lamellipodium tip triggers membrane protrusion
The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.