Early Conformational Changes Research Articles

Glu1022.53-Mediated Early Conformational Changes in the Process of Light-Induced Green Cone Pigment Activation.

Ligand-triggered activation of G protein-coupled receptors (GPCRs) relies on the phenomenon of loose allosteric coupling, which involves conformational alterations spanning from the extracellular ligand-binding domain to the cytoplasmic region, where interactions with G proteins occur. During the GPCR activation process, several intermediate and equilibrium states orchestrate the movement of the flexible and rigid transmembrane (TM) segments of the GPCR. Monitoring early conformational changes is important in unraveling the structural intricacies of the loose allosteric coupling. Here, we focus on the lumi intermediate formed by thermal relaxation from the initial photointermediate, batho in primate green cone pigment (MG), a light-sensitive GPCR responsible for color vision. Our findings from light-induced Fourier transform infrared difference spectroscopy reveal its similarity with rhodopsin, which mediates twilight vision, specifically involving the flip motion of the β-ionone ring, the relaxation of the torsional structure of the retinal, and local perturbations in the α-helix upon lumi intermediate formation. Conversely, we observe a hydrogen bond modification specific to MG's protonated carboxylic acid, identifying its origin as Glu1022.53 situated in TM2. The weakening of the hydrogen bond strength at Glu1022.53 during the transition from the batho to the lumi intermediates corresponds to a slight outward movement of TM2. Additionally, within the X-ray crystal structure of the rhodopsin lumi intermediate, we note the relocation of the Met862.53 side chain in TM2, expanding the volume of the retinal binding pocket. Consequently, the position of 2.53 emerges as the early step in the conformational shift toward light-induced activation. Moreover, given the prevalence of IR-insensitive hydrophobic amino acids at position 2.53 in many rhodopsin-like GPCRs, including rhodopsin, the hydrogen bond alteration in the C═O stretching band at Glu1022.53 of MG can be used as a probe for tracing conformational changes during the GPCR activation process.

The role of α-sheet structure in amyloidogenesis: characterization and implications.

Amyloid diseases are linked to protein misfolding whereby the amyloidogenic protein undergoes a conformational change, aggregates and eventually forms amyloid fibrils. While the amyloid fibrils and plaques are hallmarks of these diseases, they typically form late in the disease process and do not correlate with disease. Instead, there is growing evidence that smaller, soluble toxic oligomers form prior and appear to be early triggers of the molecular pathology underlying these diseases. Nearly 20 years ago, we proposed the α-sheet hypothesis after discovering that the early conformational changes observed during atomistic molecular dynamics simulations involve the formation of a non-standard protein structure, α-sheet. Furthermore, we proposed that toxic oligomers contain α-sheet structure and that preferentially targeting this structure could neutralize the toxicity, prevent further aggregation and serve as the basis for early detection of disease. Here, we present the origin of the α-sheet hypothesis and describe α-sheet structure and the corresponding mechanisms of conversion. We discuss experimental studies demonstrating that both mammalian and bacterial amyloid systems form α-sheet oligomers before converting to conventional β-sheet fibrils. Furthermore, we show that the process can be inhibited with de novo designed α-sheet peptides complementary to the structure in the toxic oligomers.

In Search of Synergistic Insect Repellents: Modeling of Muscarinic GPCR Interactions with Classical and Bitopic Photoactive Ligands.

Insect vector-borne diseases pose serious health problems, so there is a high demand for efficient molecules that could reduce transmission. Using molecular docking and molecular dynamics (MD) simulation, we studied a series of compounds acting on human and insect muscarinic acetylcholine receptors (mAChRs), a novel target of synergistic agents in pest control. We characterized early conformational changes of human M1 and fruit fly type-A mAChR G protein-coupled receptors (GPCRs) in response to DEET, IR3535, and muscarine binding based on the MD analysis of the activation microswitches known to form the signal transduction pathway in class A GPCRs. We indicated groups of microswitches that are the most affected by the presence of a ligand. Moreover, to increase selectivity towards insects, we proposed a new, bitopic, photoswitchable mAChR ligand—BQCA-azo-IR353 and studied its interactions with both receptors. Modeling data showed that using a bitopic ligand may be a promising strategy in the search for better insect control.

The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activation.

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit.

Structural and Functional Characterization of Covalently Modified Proteins Formed By a Glycating Agent, Glyoxal.

Glycation, the main consequence of hyperglycemia, is one of the major perpetrators of diabetes and several other conditions, including coronary and neurodegenerative complications. Such a hyperglycemic condition is represented by a large increase in levels of various glycation end products including glyoxal, methylglyoxal, and carboxymethyl-lysine among others. These glycation end products are known to play a crucial role in diabetic complications due to their ability to covalently modify important proteins and enzymes, specifically at lysine residues (a process termed as glycation), making them non-functional. Previous studies have largely paid attention on characterization and identification of these reactive glycating agents. Structural and functional consequences of proteins affected by glycation have not yet been critically investigated. We have made a systematic investigation on the early conformational changes and functional alterations brought about by a glycating agent, glyoxal, on different proteins. We found that the early event in glycation includes an increase in hydrodynamic diameter, followed by minor structural alterations sufficient to impair enzyme activity. The study indicates the importance of glyoxal-induced early structural alteration of proteins toward the pathophysiology of hyperglycemia/diabetes and associated conditions.

Early conformational changes at tumour bed and long term response after neoadjuvant therapy in locally-advanced rectal cancer

ObjectivesTo evaluate how changes in tumour scar depth angle and thickness in the post-neoadjuvant period relate to long-term response in locally-advanced rectal cancer patients. MethodsInformed consent was obtained from all patients and institutional review board approved this retrospective study. Sixty-nine consecutive locally-advanced rectal cancer patients who underwent neoadjuvant therapy and were selected for “Watch-and-Wait” were enrolled. Two radiologists, O1 and O2, blindly and independently reviewed the 1st and 2nd post-neoadjuvant therapy pelvic MRI T2-weighted images and recorded depth angle and thickness of the tumour scar. Value changes were calculated by simple subtraction (2nd-1st). Mann-Whitney U test was employed to assess for significant differences between sustained clinical complete responders (SCR), defined as patients with pathologic complete response or clinical complete response with a minimum follow-up of 1 year; and non-sustained complete responders (non-SCR). Interobserver agreement was estimated using intraclass correlation coefficient (ICC). Data on mrTRG, DWI and endoscopy at 1st and 2nd timepoints were retrieved for comparison. ResultsIn SCR, depth angle change between 1st (med = 10 weeks after end of radiotherapy) and 2nd (med = 23 weeks after end of radiotherapy) timepoints was significantly different (O1:p = 0.004; O2:p = 0.010): the SCR group showed a depth angle reduction (O1:med=-4.45; O2:med=-2.35), whereas non-SCRs showed a depth angle increase (O1:med=+2.60; O2:med=+7.40). Also, at 2nd timepoint, SCR scars were significantly thinner both for O1 (p = 0.003; SCR:med = 7.05 mm; non-SCR:med = 9.4 mm) and O2 (p = 0.006; SCR:med = 6.45 mm; non-SCR:med = 8.2 mm). A depth angle increase >21º between 1st and 2nd timepoints and a scar thickness >10 mm at 2nd timepoint were not sensitive but were highly specific for a non-SCR (91/94 %) for both observers. Interobserver agreement was good for scar depth angle change (ICC = 0.65) and excellent for scar thickness at 2nd timepoint (ICC = 0.84). Of the retrieved data, only DWI at 2nd timepoint was discriminative (p = 0.043) providing a similar sensitivity (33 %) and a slightly lower specificity (87.5 %). ConclusionTumour scar expansion >21° between 1st and 2nd post-neoadjuvancy MRI and a scar thickness >10 mm at 2nd post-neoadjuvancy MRI may consistently indicate a non-SCR with high specificity in locally-advanced rectal cancer patients.

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Structural elements regulating the photochromicity in a cyanobacteriochrome

The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) carrying a phycocyanobilin chromophore could be solved in both 15-Z dark-adapted state, Pr, λmax = 649 nm, and 15-E photoproduct, Pg, λmax = 536 nm (resolution, 1.6 and 1.86 Å, respectively). The structural data allowed identifying the large spectral shift of the Pr-to-Pg conversion as resulting from an out-of-plane rotation of the chromophore's peripheral rings and an outward movement of a short helix formed from a formerly unstructured loop. In addition, a third structure (2.1-Å resolution) starting from the photoproduct crystals allowed identification of elements that regulate the absorption maxima. In this peculiar form, generated during X-ray exposition, protein and chromophore conformation still resemble the photoproduct state, except for the D-ring already in 15-Z configuration and tilted out of plane akin the dark state. Due to its formation from the photoproduct, it might be considered an early conformational change initiating the parental state-recovering photocycle. The high quality and the distinct features of the three forms allowed for applying quantum-chemical calculations in the framework of multiscale modeling to rationalize the absorption maxima changes. A systematic analysis of the PCB chromophore in the presence and absence of the protein environment showed that the direct electrostatic effect is negligible on the spectral tuning. However, the protein forces the outer pyrrole rings of the chromophore to deviate from coplanarity, which is identified as the dominating factor for the color regulation.

β-Arrestin-2 BRET Biosensors Detect Different β-Arrestin-2 Conformations in Interaction with GPCRs.

β-Arrestins are critical regulators of G protein-coupled receptors (GPCRs) that desensitize G protein signaling, promote receptor internalization, and initiate signaling on their own. Recent structural findings indicate that β-arrestins adopt different conformations upon interaction with agonist-activated GPCRs. Here, we established a β-arrestin-2 conformational bioluminescence resonance energy transfer (BRET) sensor composed of the bright Nanoluc BRET donor and the red-shifted CyOFP1 BRET acceptor. The sensor monitors early intramolecular conformational changes of β-arrestin-2 in complex with a wide panel of different class A and class B GPCRs upon agonist activation and with orphan GPCRs known to spontaneously recruit β-arrestin-2. The introduction of the R170E mutant in the β-arrestin-2 sensor allowed the detection of a partially active β-arrestin-2 conformation, which is not dependent on receptor phosphorylation, while the deletion of the β-arrestin-2 finger-loop region detected the "tail-conformation" corresponding to the interaction of β-arrestin with the carboxyl-terminal domain of GPCRs. The new sensors combine the advantages of the BRET technique in terms of sensitivity, robustness, and suitability for real-time measurements with a high responsiveness toward early conformational changes to help to elucidate the different conformational states of β-arrestins associated with GPCR activation in living cells.

Drivers of α-Sheet Formation in Transthyretin under Amyloidogenic Conditions.

Amyloid diseases make up a set of fatal disorders in which proteins aggregate to form fibrils that deposit in tissues throughout the body. Amyloid-associated diseases are challenging to study because amyloid formation occurs on time scales that span several orders of magnitude and involve heterogeneous, interconverting protein conformations. The development of more effective technologies to diagnose and treat amyloid disease requires both a map of the conformations sampled during amyloidogenesis and an understanding of the molecular mechanisms that drive this process. In prior molecular dynamics simulations of amyloid proteins, we observed the formation of a nonstandard type of secondary structure, called α-sheet, that we proposed is associated with the pathogenic conformers in amyloid disease, the soluble oligomers. However, the detailed molecular interactions that drive the conversion to α-sheet remain elusive. Here we use molecular dynamics simulations to interrogate a critical event in transthyretin aggregation, the formation of aggregation-competent, monomeric species. We show that conformational changes in one of the two β-sheets in transthyretin enable solvent molecules and polar side chains to form electrostatic interactions with main-chain peptide groups to facilitate and modulate conversion to α-sheet secondary structure. Our results shed light on the early conformational changes that drive transthyretin toward the α-sheet structure associated with toxicity. Delineation of the molecular events that lead to aggregation at atomic resolution can aid strategies to target the early, critical toxic soluble oligomers.

Tau local structure shields an\xa0amyloid-forming motif and controls aggregation propensity

Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene (MAPT) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within tau’s repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306VQIVYK311. We use cross-linking mass spectrometry, recombinant protein and synthetic peptide systems, in silico modeling, and cell models to conclude that the aggregation-prone 306VQIVYK311 motif forms metastable compact structures with its upstream sequence that modulates aggregation propensity. We report that disease-associated mutations, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis.

Dynamics of the EAG1 K+ channel selectivity filter assessed by molecular dynamics simulations

EAG1 channels belong to the KCNH family of voltage gated potassium channels. They are expressed in several brain regions and increased expression is linked to certain cancer types. Recent cryo-EM structure determination finally revealed the structure of these channels in atomic detail, allowing computational investigations. In this study, we performed molecular dynamics simulations to investigate the ion binding sites and the dynamical behavior of the selectivity filter. Our simulations suggest that sites S2 and S4 form stable ion binding sites, while ions placed at sites S1 and S3 rapidly switched to sites S2 and S4. Further, ions tended to dissociate away from S0 within less than 20 ns, due to increased filter flexibility. This was followed by water influx from the extracellular side, leading to a widening of the filter in this region, and likely non-conductive filter configurations. Simulations with the inactivation-enhancing mutant Y464A or Na+ ions lead to trapped water molecules behind the SF, suggesting that these simulations captured early conformational changes linked to C-type inactivation.

Molecular assembly of lethal factor enzyme and pre-pore heptameric protective antigen in early stage of translocation.

During intoxication, the anthrax toxin lethal (LF) and edema (EF) factors initially assemble with the protective antigen (PA) on the plasma membrane of cells expressing the membrane-bound surface-exposed anthrax toxin receptor (ATR). This takes place at the physiological pH prior to entering the acidic environment of the endosome. We elucidated the molecular dynamics (MD) behaviors of the three-dimensional structure of the (PA63)7LF3 complex in various conformations and analyzed the dynamical properties of the fully loaded pre-pore complex on the plasma membrane at the physiological pH. The analysis points to the interaction networks of amino acids conserved between PA63 octamer and heptamer, which are not affected during the initial stage of the LFs binding. The simulations show an asymmetrical movement of the complex domains that directly affect LFs conformations. The conformational and structural alterations of the 2β2-2β3 loops of PA subunits are associated with pore formation. The early conformational changes of the loops appear as they peel off from the domain 2 toward domain 4 of each PA subunit. The LFs unfold in 1α1 segments of their N-terminal initiating the early stage of the pre-pore formation. The results indicate instable regions within the complex and provide important clues concerning the detail of fluctuating residues of the LF-PA interface regions at the early steps of toxins translocation.

Ion mobility-mass spectrometry-based screening for inhibition of α- synuclein aggregation.

Aberrant protein folding and formation of amyloid fibrils are associated with numerous debilitating human diseases, for which there are currently no suitable therapeutic treatments. For instance, Parkinson's disease is characterised pathologically by the intraneural accumulation of the amyloid protein α- synuclein. In order to search for new therapeutic agents that are effective in preventing the early conformational changes that precede protein aggregation, it is necessary to devise new analytical screening approaches. Here we demonstrate the use of ion mobility-mass spectrometry for screening of molecules capable of inhibiting the misfolding and aggregation of α-synuclein (specifically, the A53T human mutant). Importantly, this assay allows for the analysis of conformational changes that precede aggregation, and therefore is unique in its ability to identify inhibitors working at the earliest stages of amyloid formation. In addition, we use complementary mass spectrometry methods to probe selected protein-ligand interactions responsible for fibril inhibition.

Insights into the molecular mechanism of protein native-like aggregation upon glycation

Several human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Familial Amyloidotic Polyneuropathy, have long been associated with, structural and functional changes in disease related proteins leading to aggregation into amyloid fibrils. Such changes can be triggered by post-translational modifications. Methylglyoxal modifications have been shown to induce the formation of small and stable native-like aggregates in the case of the amyloidogenic proteins insulin and α-synuclein. However, the fundamental biophysical mechanism underlying such methylglyoxal-induced protein aggregation is not yet fully understood. In this work cytochrome c (Cyt c) was used as a model protein for the characterization of specific glycation targets and to study their impact on protein structure, stability, and ability to form native-like aggregates. Our results show that methylglyoxal covalently modifies Cyt c at a single residue and induces early conformational changes that lead to the formation of native-like aggregates. Furthermore, partially unfolded species are formed, but do not seem to be implicated in the aggregation process. This shows a clear difference from the amyloid fibril mechanisms which involve partially or totally unfolded intermediates. Equilibrium-unfolding experiments show that glycation strongly decreases Cyt c conformational stability, which is balanced with an increase of conformational stability upon aggregation. Data collected from analytical and spectroscopic techniques, along with kinetic analysis based on least-squares parameter fitting and statistical model discrimination are used to help to understand the driving force underlying glycation-induced native-like aggregation, and enable the proposal of a comprehensive thermodynamic and kinetic model for native-like aggregation of methylglyoxal glycated Cyt c.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H(I) and H(IV). Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca(2+) exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca(2+) promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca(2+) rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca(2+). Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

The Role of PIP2 in the Voltage-Dependent Activation of Kv7.1

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important regulator of ion channel activity. While PIP2 modulation of voltage-independent Kir channels has been extensively studied, the mechanism of how PIP2 potentiates the activity of voltage-dependent ion channels remains uknown. In this work, we study PIP2 activation of a voltage-dependent potassium channel (Kv7.1). Kv7.1 channels have a canonical Kv structure with a central pore-gate domain (PGD) surrounded by four peripheral voltage-sensing domains (VSDs). Kv channel activation is thought to involve two general steps. First, membrane depolarization is sensed by gating charges in the VSDs resulting in conformational changes within the VSDs from the resting state to the activated state. After all four VSDs have been activated, the second general step in Kv activation involves a concerted motion during which the PGD opens to allow ion permeation. In this study we ask if PIP2 regulates activation of Kv7.1 by potentiating the early VSD conformational changes, the concerted opening of the PGD, or both. Using the voltage-clamp fluorometry (VCF) technique to assay local conformation changes in the VSDs and the PGD simultaneously, we are able to show that although depletion of PIP2 eliminates the ionic current, the early conformational changes in the VSD do not require PIP2. This result indicates that the later conformational changes that couple VSD activation to the opening of the PGD are eliminated by PIP2 depletion. We continue this work by dissecting the molecular details of why PIP2 is required for opening of the PGD in response to VSD activation. These results may provide insights on the common principle of how Kv channels are modulated by PIP2.

Hetero-oligomerization of Neuronal Glutamate Transporters

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.

Influence of percentage of guanine molecules, OH radicals, UV irradiation and temperature on electrooxidation of short synthetic oligonucleotides

The electrooxidation of short synthetic 20-nucleotides DNA sequences with various amount of guanine molecules has been studied in a wide temperature range by square wave voltammetry and the results were compared with UV-vis and CD spectra. A twofold increase of dsDNA voltammetric peak, related to an increase in the number of electrons transferred in the guanine electrooxidation process was found to begin at a temperature lower by circa 20 °C compared to the well known increase of the dsDNA absorbance upon denaturation. Since the dsDNA voltammetric peaks are related directly to the electrooxidation of guanine and adenine, early conformational changes in dsDNA are responsible for this effect. An increase in percentage of guanine in the DNA chains caused a delay in the conformational, predenaturation changes. An exception to this behavior was found for polyguanine (100% guanine). Interestingly, two distinct ranges of change in ellipticity in the CD spectra correlate well with the changes obtained by voltammetry. We have also checked the influence of OH radicals and UV irradiation on the dsDNA oxidation.

Tracking G-protein-coupled receptor activation using genetically encoded infrared probes

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.