Buried Tryptophan Research Articles

19F NMR relaxation of buried tryptophan side chains suggest anisotropic rotational diffusion of the protein RfaH.

The recent application of 19F NMR in the study of biomolecular structure and dynamics has made it a potentially attractive probe to complement traditional 15N/13C labelled probes for backbone and sidechain dynamics, albeit with some complications. The utility of 15N relaxation rates of rigid backbone amide groups to determine the rotational diffusion tensor of proteins is well established. Here we show that the measured 19F relaxation rates of two buried and possibly immobile 19F labelled tryptophan sidechains for the multidomain protein RfaH, in its closed conformation, are in reasonable agreement with the calculated values, only when anisotropic rotational diffusion of the protein is considered. While the sparsity of 19F relaxation data from a limited number of probes precludes the experimental determination of the rotational diffusion tensor here, these results demonstrate the influence of rotational diffusion anisotropy of proteins on 19F NMR relaxation of rigid tryptophan sidechains, while adding to the expanding literature of 19F NMR relaxation data sets in biomolecules.

Triplet state spectroscopy reveals involvement of the buried tryptophan residue 310 in Glyceraldehyde-3-phosphate dehydrogenase (GAPD) in the interaction with acrylamide

Using conventional steady state and time resolved fluorescence study of the interaction between a multi-tryptophan protein and a quencher, it is difficult, if not impossible to identify the particular tryptophan residue/residues involved in the interaction. In this work we have exemplified the above contention using a multi-tryptophan protein, Glyceraldehyde-3-phosphate dehydrogenase (GAPD) from rabbit muscle having three tryptophan (Trp) residues at positions 84, 193 and 310 and a neutral quencher acrylamide in Tris buffer of pH 7.5. From the steady state and time resolved fluorescence quenching (at 298 K) with acrylamide Ksv, K and kq for the system have been calculated. Low temperature phosphorescence (LTP) spectra at 77 K of GAPD in suitable cryosolvent is known to exhibit two (0,0) bands corresponding to two tryptophan residues 193 and 310. Using the LTP study of free GAPD and GAPD – acrylamide it is possible to identify that the buried Trp 310 residue is specifically involved in the interaction with acrylamide. This is possible without doing any site-directed mutagenesis of GAPD which contains Trp residues at 84, 193 and 310. Tyrosine 320 is also specifically quenched. The results have been corroborated using the molecular docking studies. Molecular Dynamics simulation supports our contention of the involvement of Trp 310 and also shows that the other nearest residues of acrylamide are Val175 and Val232.

Dichlorophenylpyridine-Based Molecules Inhibit Furin through an Induced-Fit Mechanism.

Inhibitors of the proprotein convertase furin might serve as broad-spectrum antiviral therapeutics. High cellular potency and antiviral activity against acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported for (3,5-dichlorophenyl)pyridine-derived furin inhibitors. Here we characterized the binding mechanism of this inhibitor class using structural, biophysical, and biochemical methods. We established a MALDI-TOF-MS-based furin activity assay, determined IC50 values, and solved X-ray structures of (3,5-dichlorophenyl)pyridine-derived compounds in complex with furin. The inhibitors induced a substantial conformational rearrangement of the active-site cleft by exposing a central buried tryptophan residue. These changes formed an extended hydrophobic surface patch where the 3,5-dichlorophenyl moiety of the inhibitors was inserted into a newly formed binding pocket. Consistent with these structural rearrangements, we observed slow off-rate binding kinetics and strong structural stabilization in surface plasmon resonance and differential scanning fluorimetry experiments, respectively. The discovered furin conformation offers new opportunities for structure-based drug discovery.

Magnetization dynamics of the material with spin triplet states under the action of a weak varying field and spin-lattice interaction in zero constant field

The anisotropic dynamics of spin triplet states (STS) of molecular single crystals in zero constant and weak varying magnetic fields (weakness means the absence of saturation at the steady state and of the nutation at the pulse EPR) directed along the molecular axes, is analytically investigated. The equations are derived for the free motion of the sample magnetization, describing its linear oscillations along that molecular axis, along which its nonzero initial value was created. The tensor of the steady-state dynamical susceptibility to the varying field is found. The result of the action of a short MW pulse on STS is analytically described, containing a periodic dependence on the pulse duration and its detuning. Abovementioned results can be applied also to I=1 NMR, what is important for nitrogen-containing explosives and narcotics monitoring. The anisotropic dynamics of electron spin-lattice relaxation (SLR) at its one-phonon mechanism is investigated without the high temperature approximation over the phonon temperature; the SLR rates of the separate transitions of STS are calculated; the corresponding SLR probabilities are written in the form, which supposes the fractal dimensionality d of a lattice; the results with d=4/3 agree well with the experimental data in STS of the buried tryptophan of ribonuclease T1.

Biophysical characterization of a recombinant lipase KV1 from Acinetobacter haemolyticus in relation to pH and temperature

Spectroscopic and calorimetric methods were employed to assess the stability and the folding aspect of a novel recombinant alkaline-stable lipase KV1 from Acinetobacter haemolyticus under varying pH and temperature. Data on far ultraviolet-circular dichroism of recombinant lipase KV1 under two alkaline conditions (pH 8.0 and 12.0) at 40 °C reveal strong negative ellipticities at 208, 217, 222 nm, implying its secondary structure belonging to a α + β class with 47.3 and 39.0% ellipticity, respectively. Results demonstrate that lipase KV1 adopts its most stable conformation at pH 8.0 and 40 °C. Conversely, the protein assumes a random coil structure at pH 4.0 and 80 °C, evident from a strong negative peak at ∼ 200 nm. This blue shift suggests a general decline in enzyme activity in conjunction with the partially or fully unfolded state that invariably exposed more hydrophobic surfaces of the lipase protein. The maximum emission at ∼335 nm for pH 8.0 and 40 °C indicates the adoption of a favorable protein conformation with a high number of buried tryptophan residues, reducing solvent exposure. Appearance of an intense Amide I absorption band at pH 8.0 corroborates an intact secondary structure. A lower enthalpy value for pH 4.0 over pH 8.0 and 12.0 in the differential scanning calorimetric data corroborates the stability of the lipase at alkaline conditions, while a low Km (0.68 ± 0.03 mM) for tributyrin verifies the high affinity of lipase KV1 for the substrate. The data, herein offer useful insights into future structure-based tunable catalytic activity of lipase KV1.

Microwave-radiation-induced molecular structural rearrangement of hen egg-white lysozyme.

We have investigated the nonthermal effect of 10GHz/22 dBm microwave radiation on hen egg-white lysozyme (HEWL) over different irradiation times, ranging from 2 min to 1 h. To ensure a control over the radiation parameters, a pair of microwave rectangular waveguides is used to irradiate the samples. Optical spectroscopic measurements, which include UV-visible absorption spectroscopy, Raman spectroscopy, and far UV CD spectroscopy, reveal the exposure of the buried tryptophan (Trp) residues of the native molecule between 15 and 30 min of radiation. The higher duration of the perturbation leads to a compact structure of the protein and Trp residues are buried again. Interestingly, we do not find any change in the secondary structure of the protein even for 1 h duration of radiation. The relaxation dynamics of the irradiated molecules also has been discussed. We have shown that the molecules relax to their native configuration in 7-8 h after the radiation field is turned off. The structural rearrangement over the above timescale has further been probed by a model calculation, based on a modified Langevin equation. Our coarse-grained simulation approach utilizes the mean of atomic positions and net atomic charge of each amino acid of native HEWL to mimic the initial conformation of the molecule. The modified positions of the residues are then calculated for the given force fields. The simulation results reveal the nonmonotonous change in overall size of the molecule, as observed experimentally. The radiation parameters used in our experiments are very similar to those of some of the electronic devices we often come across. Thus, we believe that the results of our studies on a simple protein structure may help us in understanding the effect of radiation on complex biological systems as well.

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43.

The N-terminal domain of the RNA binding protein TDP-43 (NTD) is essential to both physiology and proteinopathy; however, elucidation of its folding/unfolding still remains a major quest. In this study, we have investigated the biophysical behavior of intermediate ensembles employing all-atom molecular dynamics simulations in 8 M urea accelerated with high temperatures to achieve unfolded states in a confined computation time. The cumulative results of the 2.75 μs simulations show that unfolding of the NTD at 350 K evolves through different stable and meta-stable intermediate states. The free-energy landscape reveals two meta-stable intermediates (IN and IU) stabilized by non-native interactions, which are largely hydrophilic and highly energetically frustrated. A single buried tryptophan residue, W80, undergoes solvent exposure to different extents during unfolding; this suggests a structurally heterogeneous population of intermediate ensembles. Furthermore, the structure properties of the IN state show a resemblance to the molten globule (MG) state with most of the secondary structures intact. The unfolding of the NTD is initiated by the loss of β-strands, and the unfolded (U) states exhibit a population of non-native α-helices. These non-native unfolded intermediate ensembles may mediate protein oligomerization, leading to the formation of pathological, irreversible aggregates, characteristics of disease pathogenesis.

Mapping the Long-Range Electron Transfer Route in Ligninolytic Peroxidases.

Combining a computational analysis with site-directed mutagenesis, we have studied the long-range electron transfer pathway in versatile and lignin peroxidases, two enzymes of biotechnological interest that play a key role for fungal degradation of the bulky lignin molecule in plant biomass. The in silico study established two possible electron transfer routes starting at the surface tryptophan residue previously identified as responsible for oxidation of the bulky lignin polymer. Moreover, in both enzymes, a second buried tryptophan residue appears as a top electron transfer carrier, indicating the prevalence of one pathway. Site-directed mutagenesis of versatile peroxidase (from Pleurotus eryngii) allowed us to corroborate the computational analysis and the role played by the buried tryptophan (Trp244) and a neighbor phenylalanine residue (Phe198), together with the surface tryptophan, in the electron transfer. These three aromatic residues are highly conserved in all the sequences analyzed (up to a total of 169). The importance of the surface (Trp171) and buried (Trp251) tryptophan residues in lignin peroxidase has been also confirmed by directed mutagenesis of the Phanerochaete chrysosporium enzyme. Overall, the combined procedure identifies analogous electron transfer pathways in the long-range oxidation mechanism for both ligninolytic peroxidases, constituting a good example of how computational analysis avoids making extensive trial-error mutagenic experiments.

Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The βγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization.

Aggregation of Trp>Glu Mutants of the Human Gamma-D Crystallin: A Model for Hereditary or UV-Induced Cataract

Cataract is a protein misfolding disorder resulting from formation of light-scattering aggregates of proteins from the crystallin family. These long-lived proteins account for 90% of all protein in the human eye lens. The γD crystallin consists of two symmetrical domains, each of which has a duplicated Greek key fold. Maintaining this topologically complex native fold of the γD crystallin is essential for lens transparency. A number of point mutants in the γD crystallin gene cause early-onset cataract. UV-B exposure accelerates cataract onset, and UV irradiation of purified wild-type γD results in aggregation in vitro. A distinctive feature of the Greek key fold is the presence of highly conserved buried tryptophans. Replacements of tryptophan by charged glutamate groups may represent a model of UV-induced photodamage -- introduction of a charged group into the hydrophobic core generating “denaturation from within.” We show that such Trp>Glu mutants can display vastly increased aggregation propensity under physiological conditions in vitro. Furthermore, a striking property of these mutants is their ability to drive the wild-type protein into the aggregated state. Domain swapping mechanisms can account for this aggregation behavior.

Characterization of the chandipura virus leader RNA–phosphoprotein interaction using single tryptophan mutants and its detection in viral infected cells

The phosphoprotein (P protein) of the chandipura virus (CHPV), a negative strand RNA virus, is involved in both transcription and replication of the viral life cycle. Interaction between the P protein and the viral leader (le) RNA under in vitro conditions has been previously reported for CHPV and other negative strand RNA viruses such as the rinderpest virus (RPV). However, till date, the region of the P protein involved in le RNA binding remains undefined. Moreover, the in vivo occurrence of this interaction has not been studied before. Here, we have characterised the P protein-le RNA interaction, using single tryptophan mutants of the P protein. The CHPV P protein contains two tryptophan residues located at amino acid position 105 and 135 respectively. Our previous study showed that Trp 135 is located in a buried region within a less polar environment whereas Trp 105 is more solvent-exposed. In this study we have used steady state and time resolved fluorescence spectroscopy at 298 K to show that the buried tryptophan (Trp 135) is involved in the interaction with the le RNA and the more solvent exposed Trp 105 is only slightly perturbed during this interaction. We also show that Trp 135 is responsible for the dimerization of the CHPV P protein. In addition, we have been able to demonstrate for the first time that the P protein-le RNA interaction is detectable in CHPV-infected Vero-76 cells and this interaction is augmented during the replication phase of the viral cycle.

Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a β-glycosidase enzyme (β-gly) and W492G in a β-glucuronidase enzyme (β-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of β-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of β-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate β-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.

Structure and Dynamics of Molten Globular Intermediates Encountered during the Unfolding of Barstar

The modelling of the folding and unfolding reactions of small proteins as two-state processes has provided us wealth of information on the kinetics and thermodynamics of these processes and the stability and pathway of folding. Hidden in this misleading simplicity are various intermediate states including the molten globular (MG) forms. The nature of solvation of MG forms has acquired considerable importance in the context of identifying the forces that drive protein folding and unfolding reactions.In this work we monitored the solvent accessibility during the unfolding of barstar, a small single domain protein, by following the bimolecular quenching constant (kq) associated with dynamic fluorescence quenching of the single and buried tryptophan (Trp-53) by either acrylamide or potassium iodide. A pico-second time-domain fluorimeter capable of acquiring data every 200 ms of unfolding was used. Analysis of the time-dependence of kq showed the presence of an intermediate with solvent accessibility very similar to that of the unfolded (U) state. However, structural indicators such as steady-state fluorescence intensity and time-resolved anisotropy of Trp-53 and near UV-CD indicated the presence of another intermediate within ∼ 10 ms, the zero time associated with the initiation of unfolding. Taken together, our data is consistent with the following model for the process of unfolding. N a DMG a WMG a U, where DMG and WMG refer to ‘dry’ and ‘wet’ MG forms, respectively. The demonstration of the presence of a DMG has strong implications in dissecting the overall process of folding and unfolding into individual steps, apart from revealing the forces that lead to protein folding.

Denaturation Mechanism of BSA by Urea Derivatives: Evidence for Hydrogen-Bonding Mode from Fluorescence Tools

Urea and alkyl urea derivatives, which posses a free N-H moiety in the urea molecular framework is responsible for the fluorescence quenching of BSA. Fluorescence quenching accompanied with a blue initially and subsequently a red shift in the emission maximum of BSA is observed on the addition of urea derivatives containing N-H moieties. On the contrary, a fluorescence enhancement accompanied with a shift in the emission maximum towards the blue region is observed on the addition of tetramethylurea (TMU). Urea derivatives, which posses a free N-H moiety acts as a perfect denaturant by direct hydrogen-bonding interaction with BSA resulting in the unfolding process. The unfolding of the buried tryptophan moieties to the aqueous phase does not occur, when all the N-H moieties in the urea are methyl substituted (TMU). Fluorescence spectral techniques reveal that the direct hydrogen-bonding interaction of the N-H moiety of urea molecular framework with the carbonyl oxygen moieties of BSA results in the unfolding of the tryptophan moieties to the aqueous phase, while that of the carbonyl oxygen of urea with the N-H moieties of BSA is definitely not involved in the denaturation process. Steady state and time-resolved fluorescence studies illustrate that the extent of protein folding occurs at a relatively lower concentration of unsymmetrical alkyl urea derivatives (butyl urea (BU) and ethyl urea (EU)), compared to that of urea.

Luminescence from Extrinsic and Intrinsic Probes in Solid State Amorphous Human Serum Albumin Demonstrates Solvent-Protein Slaving

The physical properties of amorphous biomolecules are important to the stability of low-moisture foods and pharmaceuticals. Freeze-dried proteins are often stabilized via inclusion of excipients. The effect on protein dynamics of substitution of surface water with sugars is unclear. To explore this question, we have conducted luminescence studies on human serum albumin (HSA) in the dry amorphous state using both extrinsic probes and intrinsic tryptophan. Phosphorescence is an ideal approach, as the long-lived triplet state of molecular probes is sensitive to the long time-scale motions of dry proteins. HSA binds luminescence probes that report on the protein's surface; it contains a single, buried tryptophan that reports on the interior of the protein. Amorphous protein-sugar films were prepared by spreading concentrated solutions of sugar + HSA with bound probe onto quartz slides, followed by rapid drying and extensive desiccation. We have bound the water-sensitive probe pyranine to HSA and measured its fluorescence spectra to extract information on the amount of the water in the protein's hydration shell. We have also made dry films from sugars + HSA bound with erythrosin B or vanillin and collected these probe's phosphorescence decays as a function of temperature. These measurements are compared to those of HSA's buried tryptophan residue. Intensity decays were fit with multi-exponential functions and the rates of non-radiative decay (kNR) were calculated from the average lifetime; kNR is dependent on the microviscosity of the site and is thus a measure of local molecular mobility. The degree to which the fits are non-exponential reveals dynamic heterogeneity experienced by the probe. Analysis of kNR Arrhenius plots reveal relations between the mobility in the bulk solid solvent with that at the surface and in the interior of the protein.

Partially Folded Aggregation Intermediates of Human γD-, γC-, and γS-Crystallin Are Recognized and Bound by Human αB-Crystallin Chaperone

Human γ-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone α-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human γ-crystallin substrate species recognized by human α-crystallin. The three major human lens monomeric γ-crystallins, γD, γC, and γS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human αB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human γ-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The αB-crystallin oligomers formed long-lived stable complexes with their γD-crystallin substrates. Using α-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone–substrate complex. Binding of substrate γ-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with α-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.

Calmodulin Disrupts the Structure of the HIV-1 MA Protein

The MA protein from HIV-1 is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. To achieve its diverse tasks, MA interacts with host cell proteins and it has been reported that one of these is the ubiquitous calcium-sensing calmodulin (CaM), which is up-regulated upon HIV-1 infection. The nature of the CaM–MA interaction has been the subject of structural studies, using peptides based on the MA sequence, that have led to conflicting conclusions. The results presented here show that CaM binds intact MA with 1:1 stoichiometry in a Ca2+-dependent manner and that the complex adopts a highly extended conformation in solution as revealed by small-angle X-ray scattering. Alterations in tryptophan fluorescence suggest that the two buried tryptophans (W16 and W36) located in the first two alpha-helices of MA mediate the CaM interaction. Major chemical shift changes occur in the NMR spectrum of MA upon complex formation, whereas chemical shift changes in the CaM spectrum are quite modest and are assigned to residues within the normal target protein-binding hydrophobic clefts of CaM. The NMR data indicate that CaM binds MA via its N- and C-terminal lobes and induces a dramatic conformational change involving a significant loss of secondary and tertiary structure within MA. Circular dichroism experiments suggest that MA loses ∼20% of its α-helical content upon CaM binding. Thus, CaM binding is expected to impact upon the accessibility of interaction sites within MA that are involved in its various functions.

Phosphorescence from Single Tryptophan in Amorphous Solid Human Serum Albumin Exhibits Solvent-Protein Dynamics Slaving

The physical properties of amorphous biomolecules are important to the texture and stability of low-moisture foods, the stability of pharmaceuticals, the permeability of edible films, and the viability of organisms during anhydrobiosis. Protein stability is often improved via the inclusion of small-molecule excipients during freeze-drying and organisms overproduce sugars such as sucrose or trehalose during anhydrobiosis. The effect on internal protein dynamics caused by substitution of a protein's surface water molecules with small sugar molecules is unclear. To explore this question, we have analyzed tryptophan phosphorescence decays of human serum albumin (HSA) in the dry amorphous solid state. Phosphorescence is an ideal approach, as the long-lived triplet state of tryptophan is sensitive to the long time-scale molecular motions of proteins in the dry state. Human serum albumin (HSA) was chosen because it contains a single, buried tryptophan residue and thus can provide information on the local dynamics of a specific site in the interior of the protein. Amorphous protein films were prepared by spreading concentrated solutions of HSA with and without sugar onto quartz slides, followed by rapid drying and extensive desiccation. Phosphorescence intensity decays were collected and fit with multiple exponential functions. From the average lifetime of these fits the rates of non-radiative decay (kNR) of the triplet state were calculated; kNR is dependent on the microviscosity of the site and is thus a measure of molecular mobility of the HSA tryptophan site. At all temperatures this measure of molecular mobility was lower in the films containing sucrose. Break-point analysis of a kNR Arrhenius plot revealed two temperature regimes with a transition occurring at the glass transition temperature of sucrose. Research supported in part by the National Research Initiative of USDA-CSREES.

Structural and Electronic Insights into Tryptophan Radicals in Proteins

Tryptophan radicals play significant roles in mediating biological electron transfer reactions and catalytic processes. Despite their prevalence in Nature, there is a dearth of knowledge on the structure and dynamics of these transient species. Here, we report absorption, EPR, and resonance Raman spectra of long-lived solvent-exposed and buried tryptophan neutral radicals in azurin mutants. These spectra reveal important markers and trends that reflect the local environment, structure, hydrogen bonding state, and protonation state of the radical; comparison of these spectra to those of the closed-shell counterparts indicates that the spectral features of the radical are highly sensitive to structure and environment. The results and analysis described here not only provide detailed basis spectra for ongoing work, but also shed light on the nature of proton-coupled electron transfer reactions.

The effect of the cosolvent trifluoroethanol on a tryptophan side chain orientation in the hydrophobic core of troponin C

The unique biophysical properties of tryptophan residues have been exploited for decades to monitor protein structure and dynamics using a variety of spectroscopic techniques, such as fluorescence and nuclear magnetic resonance (NMR). We recently designed a tryptophan mutant in the regulatory N-domain of cardiac troponin C (F77W-cNTnC) to study the domain orientation of troponin C in muscle fibers using solid-state NMR. In our previous study, we determined the NMR structure of calcium-saturated mutant F77W-V82A-cNTnC in the presence of 19% 2,2,2-trifluoroethanol (TFE). TFE is a widely used cosolvent in the biophysical characterization of the solution structures of peptides and proteins. It is generally assumed that the structures are unchanged in the presence of cosolvents at relatively low concentrations, and this has been verified for TFE at the level of the overall secondary and tertiary structure for several calcium regulatory proteins. Here, we present the NMR solution structure of the calcium saturated F77W-cNTnC in presence of its biological binding partner troponin I peptide (cTnI(144-163)) and in the absence of TFE. We have also characterized a panel of six F77W-cNTnC structures in the presence and absence TFE, cTnI(144-163), and the extra mutation V82A, and used (19)F NMR to characterize the effect of TFE on the F77(5fW) analog. Our results show that although TFE did not perturb the overall protein structure, TFE did induce a change in the orientation of the indole ring of the buried tryptophan side chain from the anticipated position based upon homology with other proteins, highlighting the potential dangers of the use of cosolvents.