Changes In Tryptophan Fluorescence Research Articles

Methylglyoxal-induced modification of myoglobin: An insight into glycation mediated protein aggregation.

Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with the proteins to form advanced glycation end products (AGEs) following a Maillard-like reaction. In a time-dependent reaction study of MG with the heme protein myoglobin (Mb), MG was found to induce significant structural alterations of the heme protein, such as heme loss, changes in tryptophan fluorescence, and decrease of α-helicity with increased β-sheet content. These changes were found to occur gradually with increasing period of incubation. Incubation of Mb with MG induced the formation of several AGE adducts, including, carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87, carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139. MG induced amyloid-like aggregation of Mb was detected at a longer period of incubation. MG-derived AGEs, therefore, appear to have an important role as the precursors of protein aggregation, which, in turn, may be associated with pathophysiological complications.

Intrinsic tryptophan fluorescence quenching by iodine in non-canonical amino acid reveals alteration of the hydrogen bond network in the photoactive orange carotenoid protein

The Orange Carotenoid Protein (OCP) regulates cyanobacterial photosynthetic activity through photoactivation in intense light. A hydrogen bonding network involving the keto-carotenoid oxygen and Y201 and W288 residues prevents the spontaneous activation of dark-adapted OCP. To investigate the role of the hydrogen bonds in OCP photocycling, we introduced non-canonical amino acids near the keto-carotenoid, particularly iodine at the meta-position of Y201. This modification significantly increased the yield of red OCP photoproducts, albeit with a shorter lifetime. Changes in tryptophan fluorescence during photocycling influenced by the presence of iodine near W288 revealed interactions between Y201 and W288 in the absence of the carotenoid in the C-domain. We propose that upon the relaxation of red states, a ternary complex with the carotenoid is formed. Analysis of spectral signatures and interaction energies indicates that the specific iodo-tyrosine configuration enhances interactions between the carotenoid and W288.

Advances in Mixer Design and Detection Methods for Kinetics Studies of Macromolecular Folding and Binding on the Microsecond Time Scale.

Many important biological processes such as protein folding and ligand binding are too fast to be fully resolved using conventional stopped-flow techniques. Although advances in mixer design and detection methods have provided access to the microsecond time regime, there is room for improvement in terms of temporal resolution and sensitivity. To address this need, we developed a continuous-flow mixing instrument with a dead time of 12 to 27 µs (depending on solution viscosity) and enhanced sensitivity, sufficient for monitoring tryptophan or tyrosine fluorescence changes at fluorophore concentrations as low as 1 µM. Relying on commercially available laser microfabrication services, we obtained an integrated mixer/flow-cell assembly on a quartz chip, based on a cross-channel configuration with channel dimensions and geometry designed to minimize backpressure. By gradually increasing the width of the observation channel downstream from the mixing region, we are able to monitor a reaction progress time window ranging from ~10 µs out to ~3 ms. By combining a solid-state UV laser with a Galvano-mirror scanning strategy, we achieved highly efficient and uniform fluorescence excitation along the flow channel. Examples of applications, including refolding of acid-denatured cytochrome c triggered by a pH jump and binding of a peptide ligand to a PDZ domain, demonstrate the capability of the technique to resolve fluorescence changes down to the 10 µs time regime on modest amounts of reagents.

Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.

Investigation of structural factors controlling loop dynamics in acyl protein thioesterases

Acyl protein thioesterases catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. Recently, human APTs were shown to use a two‐step catalytic mechanism involving initial membrane binding of an exposed flexible loop followed by substrate binding and cleavage. This two‐step catalytic mechanism is likely conserved as a bacterial homologue of human APTs is also regulated by the large‐scale movement of an essential dynamic loop upon membrane binding. However, the causes for the movement of this dynamic loop in APTs are unknown. Herein, we investigated factors that trigger loop movement in human and bacterial APTs. In particular, APTs were assayed with inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence of a central tryptophan on the dynamic loop before and after exposure to these conditions. Although inhibitors and substrate mimics induced minor changes in loop dynamics, larger shifts were measured with various membrane‐mimicking surfactants, suggesting that APT loop dynamics are mainly regulated by proximity to the plasma membrane. Ultimately, understanding the factors controlling APT function could provide novel mechanisms for controlling cell signaling.

Screening of novel excipients for freeze-dried protein formulations

The typical excipients used as bulking agents and lyoprotectants for freeze-drying are usually limited to only a few selected substances, such as sucrose and mannitol. Considering the sheer diversity amongst proteins, it is doubtful that this limited choice should, in every case, provide the best possible option in order to achieve the most stable product. In this work, a screening of 12 proteins with 64 excipients was conducted in order to increase the knowledge space of potential excipients. Three critical quality attributes (CQAs) of the freeze-dried products, namely the solid state, the cake appearance and the protein integrity based on changes in tryptophan fluorescence were investigated by high throughput X-ray powder diffraction, image analysis and intrinsic fluorescence spectroscopy, respectively. It was found, that in some cases the excipient had a dominating influence on the CQAs, whilst in other cases the CQAs were primarily protein dependent, or that the CQAs were dependent on the combination of both. In the course of this investigation, a general view of potentially relevant excipients, and their interplay with various proteins, was obtained, thereby furthermore paving the way for the use of novel freeze-drying excipients.

Fragments of Locusta migratoria apoLp-III provide insight into lipid binding

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein with a critical role in lipid transport in insects. The protein is composed of a bundle of five amphipathic α-helices which undergo a large conformational change upon lipid binding. To better understand the apoLp-III lipid binding interaction, the protein was cleaved by cyanogen bromide upon introduction of a S92M mutation, generating an N-terminal fragment corresponding to the first three helices (NTH1-3) and a C-terminal fragment of the last two helices (CTH4-5). MALDI-TOF analysis of the HPLC purified fragments provided masses of 9863.8 Da for NTH1-3 and 7497.0 Da for CTH4-5 demonstrating that the intended fragments were obtained. Circular dichroism spectra revealed a decrease in helical content from 82% for the intact protein to 57% for NTH1-3 and 41% for CTH4-5. The fragments adopted considerably higher α-helical structure in the presence of trifluoroethanol or phospholipids. Equimolar mixing of the two fragments did not result in changes in helical content or tryptophan fluorescence, indicating recombination into the native protein fold did not occur. The rate of protein induced dimyristoylphosphatidylcholine vesicle solubilization increased 15-fold for NTH1-3 and 100-fold for CTH4-5 compared to the intact protein. Despite the high activity in phospholipid vesicle interaction, CTH4-5 did not protect phospholipase-treated low-density lipoprotein from aggregation. In contrast, NTH1-3 provided protection to lipoprotein aggregation similar to the intact protein, indicating that specific amino acid residues in this part of apoLp-III are essential for lipoprotein binding interaction.

Investigation of Factors that Trigger Essential Conformational Changes in FTT258

FTT258 is a close bacterial relative of human acyl protein thioesterase (hAPT), a protein linked to cancer. hAPT removes long chain lipids from key cancer signaling proteins. Removal of these lipids requires their insertion into a pocket within hAPT, which is only exposed after the movement of a flexible loop, a common feature between hAPT and FTT258. However, the causes for the movement of this loop are unknown. I investigated factors that may trigger loop movement in APTs like hAPT and FTT258. In particular, I exposed FTT258 to inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants in order to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence before and after exposure to these conditions. Ultimately, understanding the factors controlling FTT258 and hAPT function can provide novel mechanisms for inhibiting cancer progression.Support or Funding InformationNSF MCB‐1812971

A Study of the Structure of Trypsin-Like Serine Proteinases. 2. A Study of Tryptophan Fluorescence in Variants of Miniplasminogen with an Altered Primary Structure

Abstract—Variants of miniplasminogen with an altered primary structure have been designed to study previously described changes in tryptophan fluorescence during plasminogen activation by urokinase. Miniplasminogens that contain the Trp141, Trp215, and Leu217 substitutions (the position of the amino-acid residue is specified by the Chymotrypsin nomenclature) have been tested. The activation of mutants containing a single tryptophan substitution induced changes in the fluorescence spectrum. No changes in the fluorescence spectrum were observed in the case of miniplasminogen containing a double tryptophan substitution. This indicates that both Trp141 and 215 contribute to the fluorescence spectrum shift; however, as suggested previously, the contribution of changes in the Trp215 microenvironment is greater. The Trp141, Trp215, and Leu217 amino-acid substitutions in miniplasminogen have no effect on the rate of plasminogen activation by urokinase. The replacement of the conserved Trp215 residue in serine proteases led to a significant reduction but not loss of the amidolytic activity of plasmin.

Characterization of the Assembly Dynamics of Streptococcus Pneumoniae FtsZ using Intrinsic Tryptophan Fluorescence

FtsZ is a prokaryotic cytoskeletal protein, which assembles into the Z‐ring at the division site and orchestrates the cell division process in bacteria. FtsZ has GTPase activity and self‐assembles to form polymers. We investigated the assembly characteristics of FtsZ from the pathogenic organism, Streptococcus pneumoniae. The critical concentration for FtsZ polymerization was found to be 2.4 ± 0.1 and 2.2 ± 0.2 μM measured using sedimentation assay and GTPase assay, respectively. The results indicated that the GTPase activity and assembly are closely linked to each other. We identified that FtsZ from Streptococcus species contains two native tryptophan (Trp) residues unique to this genus. Interestingly, we observed that during polymerization, the tryptophan fluorescence of FtsZ increases showing that the assembly of FtsZ can be monitored using the native tryptophan fluorescence. Using tryptophan fluorescence along with light scattering, sedimentation assay and electron microscopy we examined the effects of divalent cations, calcium and magnesium, on FtsZ assembly. Divalent magnesium enhanced the tryptophan fluorescence change during FtsZ assembly showing that it promoted the longitudinal assembly of FtsZ. Divalent calcium induced the bundling of FtsZ filaments thereby affecting the lateral interactions of FtsZ polymers but did not enhance the tryptophan fluorescence, showing the sensitivity of tryptophan fluorescence to longitudinal assembly. Next, we sought to investigate the contribution of individual tryptophan residues to fluorescence of FtsZ and use them for understanding the assembly of FtsZ. A homology model for FtsZ from Streptococcus pneumoniae showed that the two tryptophan residues were present at the C‐terminal of the protein – one at the 298th position of FtsZ near the T7 loop and the other at 378th position in the C‐terminal tail region. We used site‐directed mutagenesis to introduce point mutations to FtsZ at these positions to mutate individual tryptophan residues to cysteine or phenylalanine residues to create W294C/F and W378C/F mutant proteins. W378C/F mutants showed a similar change in tryptophan fluorescence during polymerization as of wild‐type, while W294C/F mutants did not show any change. This indicated that the Trp at the 294th position is responsible for the change in fluorescence during polymerization. This further suggested that the environment around 294W changes during the polymerization while the local environment around the 378W does not change. The two tryptophan residues in the protein allow us to use the intrinsic tryptophan fluorescence to gain new insights into FtsZ assembly. Further, it would be interesting to explore the use of these mutant proteins in following the domain movements and conformational changes occurring during the assembly and disassembly of FtsZ.Support or Funding InformationThe work is supported by a grant from Department of Science and Technology (DST, India) to D.P.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of structural factors controlling loop dynamics in acyl protein thioesterases

FTT258 is a close bacterial relative of human acyl protein thioesterases (APTs). Human APTs catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. For FTT258, catalysis was hypothesized to be regulated by the large‐scale movement of an essential dynamic loop, which transitioned the protein between active and inactive states. However, the causes for the movement of this loop are unknown. Herein, we investigated factors that trigger loop movement in APTs like FTT258. In particular, FTT258 was exposed to inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence of a central tryptophan on the dynamic loop before and after exposure to these conditions. Although inhibitors and substrate mimics induced minor changes in loop dynamics, larger shifts were measured with various membrane‐mimicking surfactants, suggesting that APT loop dynamics are mainly regulated by proximity to the plasma membrane. Ultimately, understanding the factors controlling APT function could provide novel mechanisms for controlling cell signaling.Support or Funding InformationFunded by NSF MCB‐1812971This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NAD binding by human CD38 analyzed by Trp189 fluorescence

The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca2+ mobilizing second messengers ADP-ribose (ADPR), 2′-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189. Amplitude of the fluorescence decrease was fitted as one-site binding curve revealing a dissociation constant for NAD of 29 μM. A comparable dissociation constant was found with the catalytically inactive sCD38 mutant (KD 37 μM NAD) indicating that binding of NAD is not significantly affected by the mutation. The NAD-induced decrease in Trp fluorescence completely recovered in case of sCD38. Kinetics of recovery was slowed down with decreasing temperature and sCD38 concentration and increasing NAD concentration demonstrating that recovery in fluorescence is proportional to the enzymatic activity of sCD38. Accordingly, recovery in fluorescence was not observed with the catalytically inactive mutant.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Does protein oxidation affect proteolysis in low sodium Chinese traditional bacon processing?

This work investigated protein oxidation, proteolysis and their interrelationship during the manufacturing process of low-sodium Chinese traditional bacon. The bacons were prepared by four different salting treatments: substituting sodium chloride (NaCl) using 0, 30, 50, and 70% of potassium chloride (KCl). Protein oxidation was evaluated by total carbonylation, thiol oxidation, and tryptophan fluorescence changes. Proteolysis was detected by non-protein nitrogen, free amino acid, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the manufacturing process of low-sodium Chinese bacon, protein oxidation and protein proteolysis were simultaneously observed. Electrophoresis pattern showed that 70% substitution of KCl could promote proteolysis, meanwhile, protein oxidation possibly facilitated by 70% KCl replacement resulted in reduced tryptophan fluorescence and increased total carbonyl content. Additionally, a good correlation between protein oxidation and proteolysis was observed.

Investigating Carrier Domain Positioning During Catalytic Turnover in Pyruvate Carboxylase

A central debate in protein biochemistry focuses on the mechanism by which ligand binding correlates with protein conformational change. Two primary hypotheses describe this process: the induced‐fit hypothesis states that ligand binding induces a conformational change in the protein, while the conformational ensembles hypothesis states that the protein pre‐exists in multiple conformational states, with ligand binding shifting the equilibrium towards a preferred conformation. Pyruvate carboxylase (PC) is a multi‐domain, swinging‐arm enzyme that coordinates reactions between two remote active sites and serves as a structurally and biophysically accessible paradigm to study the relationship between ligand binding and protein conformational change. PC catalyzes the ATP‐dependent carboxylation of pyruvate to oxaloacetate, using bicarbonate as the carboxyl group donor. The biotin cofactor on the biotin carboxyl carrier protein (BCCP) domain is carboxylated via a MgATP‐dependent reaction in the biotin carboxylase (BC) domain. The BCCP domain then translocates to the carboxyltransferase (CT) domain on an opposing subunit, where the carboxyl group is transferred from the biotin cofactor to pyruvate, generating oxaloacetate.In order to better understand the mechanism of carrier domain translocation, the equilibrium positioning of the BCCP domain was directly observed using site‐specific cross‐linking to trap the BCCP domain in the putative exo‐binding site on the opposing subunit. This approach permits a detailed analysis of BCCP domain positioning in response to various substrates and allosteric effectors. Samples were first analyzed using SDS‐PAGE and densitometry to measure BCCP cross‐linked dimers as reporters for equilibrium positioning changes. Given that BCCP cross‐linking inactivates the enzyme, rates of inactivation were measured to assess changes in BCCP domain positioning in the presence of substrates and effectors. Finally, intrinsic tryptophan fluorescence changes were measured on fast timescales to more accurately determine the effect of ligands on the rates of BCCP domain repositioning. These studies reveal that substrates and effectors shift the BCCP domain positioning from one domain to another and offer the first evidence that the carrier domain exists in a dynamic equilibrium between active sites.Support or Funding InformationThis work was supported by the National Institutes of Health grant GM117540.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pulsed Light Inactivation of Mushroom Polyphenol Oxidase: a Fluorometric and Spectrophotometric Study

Polyphenol oxidase (PPO) is one of the most important food enzymes; it is responsible for the browning of many foods. Pulsed light (PL) is a non-thermal method of food preservation that is able to inactivate PPO. The aim of this work was to gain insight into the mechanism of PPO inactivation by PL. To this, the kinetics of PPO inactivation by PL was measured, together with associated changes in tryptophan fluorescence, KI fluorescence quenching, and turbidity; and results were analyzed by parameter A and phase diagram methods. Enzyme inactivation followed the Weibull model. Tryptophan fluorescence decreased during PL treatment, as well as the parameter A, while Stern-Volmer constants increased and turbidity was constant. The phase diagram showed only two populated states. There was a high correlation between the loss of activity and parameter A. Results indicate that under the experimental conditions, the inactivation of PPO by PL is an all-or-none process where the enzyme progressively unfolds with no evidence of aggregation.

Search for DNA damage by human alkyladenine DNA glycosylase involves early intercalation by an aromatic residue

DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping, whereby the target nucleotide is rotated 180° into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase. Previous kinetic studies provided evidence for the formation of a transient complex that precedes the stable flipped-out complex, but it is not clear how this complex differs from nonspecific complexes. We used site-directed mutagenesis and transient-kinetic approaches to investigate the timing of Tyr162 intercalation for AAG. The tryptophan substitution (Y162W) appeared to be conservative, because the mutant protein retained a highly favorable equilibrium constant for flipping the 1,N6-ethenoadenine (ϵA) lesion, and the rate of N-glycosidic bond cleavage was identical to that of the wild-type enzyme. We assigned the tryptophan fluorescence signal from Y162W by removing two native tryptophan residues (W270A/W284A). Stopped-flow experiments then demonstrated that the change in tryptophan fluorescence of the Y162W mutant is extremely rapid upon binding to either damaged or undamaged DNA, much faster than the lesion-recognition and nucleotide flipping steps that were independently determined by monitoring the ϵA fluorescence. These observations suggest that intercalation by this aromatic residue is one of the earliest steps in the search for DNA damage and that this interaction is important for the progression of AAG from nonspecific searching to specific-recognition complexes.

Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure.

Fluorescence spectroscopy, relying on intrinsic protein fluorophores, is one of the most widely used methods for studying protein folding, protein-ligand interactions, and protein dynamics. Tryptophan is usually the fluorophore of choice, given its sensitivity to its environment and having the highest quantum yield of the natural amino acids; however, changes in tryptophan fluorescence can be difficult to interpret in terms of specific structural changes. The introduction of quenchers of tryptophan fluorescence can provide information about specific structures, particularly if quenching is short-range; however, the most commonly employed quencher is histidine, and it is effective only when the imidazole side chain is protonated, thus limiting the pH range over which this approach can be employed. In addition, histidine is not always a conservative substitution and is likely to be destabilizing if inserted into the hydrophobic core of proteins. Here we illustrate the use of a Trp-selenomethionine (MSe) pair as a specific probe of protein structure. MSe requires a close approach to Trp to quench its fluorescence, and this effect can be exploited to design specific probes of α-helix and β-sheet formation. The approach is illustrated using equilibrium and time-resolved fluorescence measurements of designed peptides and globular proteins. MSe is easily incorporated into proteins and provides a conservative replacement for hydrophobic side chains, and MSe quenching of Trp fluorescence is pH-independent. The oxidized form of MSe, selenomethionine selenoxide, is also an efficient quencher of Trp fluorescence.

Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity.

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism.

Monitoring GPCR Conformational Changes during Agonist Release in Real-Time: Evidence that Transmembrane Helix 6 (TM6) Movement in Rhodopsin Lags Behind Retinal Release

GPCR activation involves an outward movement of the cytoplasmic end of TM6, which exposes a binding site for G proteins, arrestins, and kinases. This movement, observed for several GPCRs (including rhodopsin and the β2 adrenergic receptor), appears critical for formation of the active receptor state. Previously, we showed real-time monitoring of TM6 movement using Tryptophan-Induced Quenching (TrIQ) of fluorescence of a bimane probe on TM6 by a tryptophan on TM3 (Schafer, Janz, and Farrens Biophysics Abstract 2014). The TrIQ data are consistent with an outward TM6 movement during activation; which then returns to its original position as the agonist, all-trans retinal (ATR), exits the receptor. We initially concluded that this TM6 “resetting” occurs simultaneously with agonist release, as the rates are similar to changes in intrinsic tryptophan fluorescence we measure during ATR dissociation. However, our more recent and precise measurements reveal a small, but reproducible time-lag between ATR release and the TM6 resetting. Interestingly, this lag slightly increases for a constitutively inactive mutant, as well as for another mutation inside the retinal binding pocket. Similarly, dramatically increasing the agonist dissociation rate by chemical attack (hydroxylamine) exacerbates the lag. Although the molecular mechanism underlying the delay between ATR release and TM6 movement is still unknown, the similar activation energies (∼21kcal/mol) they exhibit by Arrhenius analysis suggest a common mechanism for both. We propose these results suggest a heretofore unidentified empty opsin is present immediately after retinal release, one that differs from the “traditional” inactive opsin conformation, in which TM6 adopts an inward position. Our further investigations into this phenomenon and the implications they have on the process of retinal binding and release will be discussed along with potential caveats.