Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels.

Actin and myosin are the two main proteins required for cell motility and muscle contraction. The structure of their strongly bound complex-rigor state-is a key for delineating the functional mechanism of actomyosin motor. Current knowledge of that complex is based on models obtained from the docking of known atomic structures of actin and myosin subfragment 1 (S1; the head and neck region of myosin) into low-resolution electron microscopy electron density maps, which precludes atomic- or side-chain-level information. Here, we use radiolytic protein footprinting for global mapping of sites across the actin molecules that are impacted directly or allosterically by myosin binding to actin filaments. Fluorescence and electron paramagnetic resonance spectroscopies and cysteine actin mutants are used for independent, residue-specific probing of S1 effects on two structural elements of actin. We identify actin residue candidates involved in S1 binding and provide experimental evidence to discriminate between the regions of hydrophobic and electrostatic interactions. Focusing on the role of the DNase I binding loop (D-loop) and the W-loop residues of actin in their interactions with S1, we found that the emission properties of acrylodan and the mobility of electron paramagnetic resonance spin labels attached to cysteine mutants of these residues change strongly and in a residue-specific manner upon S1 binding, consistent with the recently proposed direct contacts of these loops with S1. As documented in this study, the direct and indirect changes on actin induced by myosin are more extensive than known until now and attest to the importance of actin dynamics to actomyosin function.

Modeling of protein binary complexes using structural mass spectrometry data.

In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints-positive and/or negative-in the docking step and are also used to decide the type of energy filter-electrostatics or desolvation-in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure.

Three-dimensional structure of cofilin bound to monomeric actin derived by structural mass spectrometry data.

The cytoskeletal protein, actin, has its structure and function regulated by cofilin. In the absence of an atomic resolution structure for the actin/cofilin complex, the mechanism of cofilin regulation is poorly understood. Theoretical studies based on the similarities of cofilin and gelsolin segment 1 proposed the cleft between subdomains 1 and 3 in actin as the cofilin binding site. We used radiolytic protein footprinting with mass spectrometry and molecular modeling to provide an atomic model of how cofilin binds to monomeric actin. Footprinting data suggest that cofilin binds to the cleft between subdomains 1 and 2 in actin and that cofilin induces further closure of the actin nucleotide cleft. Site-specific fluorescence data confirm these results. The model identifies key ionic and hydrophobic interactions at the binding interface, including hydrogen-bonding between His-87 of actin to Ser-89 of cofilin that may control the charge dependence of cofilin binding. This model and its implications fill an especially important niche in the actin field, owing to the fact that ongoing crystallization efforts of the actin/cofilin complex have so far failed. This 3D binary complex structure is derived from a combination of solution footprinting data and computational approaches and outlines a general method for determining the structure of such complexes.

Biochemical implications of a three-dimensional model of monomeric actin bound to magnesium-chelated ATP.

Actin structure is of intense interest in biology due to its importance in cell function and motility mediated by the spatial and temporal regulation of actin monomer-filament interconversions in a wide range of developmental and disease states. Despite this interest, the structure of many functionally important actin forms has eluded high-resolution analysis. Due to the propensity of actin monomers to assemble into filaments structural analysis of Mg-bound actin monomers has proven difficult, whereas high-resolution structures of actin with a diverse array of ligands that preclude polymerization have been quite successful. In this work, we provide a high-resolution structural model of the Mg-ATP-actin monomer using a combination of computational methods and experimental footprinting data that we have previously published. The key conclusion of this study is that the structure of the nucleotide binding cleft defined by subdomains 2 and 4 is essentially closed, with specific contacts between two subdomains predicted by the data.

Binding of heme to human serum albumin: steady-state fluorescence, circular dichroism and optical difference spectroscopic studies.

The binding of monomeric heme to human serum albumin (HSA) was investigated using steady-state fluorescence, circular dichroism (CD) and optical difference spectroscopic (ODS) techniques. The existence of one strong binding site for heme on HSA was confirmed by titrating heme with HSA and following the quenching of tryptophan (Trp214) fluorescence emission intensity that occurred due to energy transfer. Up to around 1:1 stoichiometric ratio of HSA/heme, the quenching was observed to be very strong, however at higher ratios the quenching progressed very weakly. Similarly, the negative CD band centered at -397 nm, which appeared on adding heme to HSA, increased in intensity on sequential addition of heme up to [heme]/[HSA] = 1. Titration of HSA with heme was followed by ODS and the dissociation constant K(D) = (4.0 +/- 1.0) x 10(-5) M was deduced. Results have been explained on the basis of Michaelis-Menton type of mechanism for the heme binding, in which heme first binds reversibly to His146 at the surface of the protein to form an intermediate complex, followed by irreversible binding to Tyr161 in the interior of the protein.

Ultrafast hydration dynamics in protein unfolding: human serum albumin.

We report studies of unfolding and ultrafast hydration dynamics of the protein human serum albumin. Unique in this study is our ability to examine different domains of the same protein and the intermediate on the way to the unfolded state. With femtosecond resolution and site-selective labeling, we isolate the dynamics of domains I and II of the native protein, domain I of the intermediate at 2 M guanidine hydrochloride, and the unfolded state at 6 M of the denaturant. For studies of unfolding, we used the fluorophores, acrylodan (covalently bound to Cys-34 in domain I) and the intrinsic tryptophan (domain II), whereas for hydration dynamics, we probed acrylodan and prodan; the latter is bound to domain II. From the time-dependent spectra and the correlation functions, we obtained the time scale of dynamically ordered water: 57 ps for the more stable domain I and 32 ps for the less stable domain II, in contrast to approximately 0.8 ps for labile, bulk-type water. This trend suggests an increased hydrophilic residues-water interaction of domain I, contrary to some packing models. In the intermediate state, which is characterized by essentially intact domain I and unfolded domain II, the dynamics of ordered water around domain I is nearly the same (61 ps) as that of native state (57 ps), whereas that in the unfolded protein is much shorter (13 ps). We discuss the role of this fluidity in the correlation between stability and function of the protein.

Activity, stability and conformational flexibility of seed coat soybean peroxidase.

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase superfamily that includes the classical peroxidase, namely horseradish peroxidase (HRP). We have measured the catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(M)) of SBP and that of HRP-C for the oxidation of ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonate)] by hydrogen peroxide at 25 degrees C. We observed that the k(cat) and k(cat)/K(M) values for SBP are much higher than those for HRP-C at all pH values, rendering SBP a more potent peroxidase. This is attributed to the relatively more solvent exposed delta-meso heme edge in SBP. We observed that the maximum catalytic activity and conformational stability of SBP is at pH approximately 5.5. A pH maximum of 5.0 for the catalytic activity of SBP has recently been reported. Estimation of secondary structural elements at various pH values indicated that there is a maximal reduction of beta-strands and beta-turns at pH 5.5 causing the heme to be further exposed to the solvent and increasing the overall conformational flexibility of the protein.

Thermal unfolding of soybean peroxidase. Appropriate high denaturant concentrations induce cooperativity allowing the correct measurement of thermodynamic parameters.

We have earlier reported that both guanidine hydrochloride (GdnHCl)-induced and heat-induced unfolding of seed coat soybean peroxidase (SBP), monitored by far UV CD, show single step transition. However, although GdnHCl-induced unfolding follows a two-state pathway, the heat-induced denaturation proceeds through intermediates as indicated by the very low cooperativity of transition. In the former case, analysis of the data based on the two-state model gives true thermodynamic parameters, whereas underestimated values are obtained in the latter case. Available complex equations also cannot be applied for the analysis of the thermal unfolding of SBP due to the absence of separate transitions for the intermediates. In the present study, we report a method to obtain true thermodynamic parameters from thermal transition curves of SBP using the two-state model. When SBP is subjected to thermal unfolding at high GdnHCl concentrations (5.8-6.9 M), cooperative behavior is observed, which allowed the analysis by the two-state model to determine their thermodynamic parameters. We then obtained the thermodynamic parameters in the absence of GdnHCl by extrapolating the graph of linear dependence of DeltaH(m) on T(m) to the T(m) corresponding to 0 m GdnHCl. Another key point for checking the validity of our method was the fact that the unfolded state of SBP generated by either heat or GdnHCl is the same by which we could cross-check our results with that obtained from GdnHCl unfolding. Having obtained the true thermodynamic parameters, we report a detailed thermodynamic study of SBP. Further we address the effect of heme in the thermal unfolding mechanism of SBP.

Thermal and conformational stability of seed coat soybean peroxidase.

Soybean peroxidase (SBP) obtained from the soybean seed coats belongs to class III of the plant peroxidase superfamily. Detailed circular dichroism and steady state fluorescence studies have been carried out to monitor thermal as well as denaturant-induced unfolding of SBP and apo-SBP. Melting of secondary and tertiary structures of SBP occurs with characteristic transition midpoints, T(m), of 86 and 83.5 degrees C, respectively, at neutral pH. Removal of heme resulted in greatly decreased thermal stability of the protein (T(m) = 38 degrees C). The deltaG degrees (H2O) determined from guanidine hydrochloride-induced denaturation at 25 degrees C and at neutral pH is 43.3 kJ mol(-1) for SBP and 9.0 kJ mol(-1) for apo-SBP. Comparison with the reported unfolding data of the homologous enzyme, horseradish peroxidase (HRP-C), showed that SBP exhibits significantly high thermal and conformational stability. We show that this enhanced structural stability of SBP relative to HRP-C arises due to the unique nature of their heme binding. A stronger heme-apoprotein affinity probably due to the interaction between Met37 and the C8 heme vinyl substituent contributes to the unusually high structural stability of SBP.

Spectroscopic studies on human serum albumin and methemalbumin: optical, steady-state, and picosecond time-resolved fluorescence studies, and kinetics of substrate oxidation by methemalbumin.

The nature of the heme environment in methemalbumin, the Fe(III) protoporphyrin IX (heme)-human serum albumin (HSA) complex, was investigated by optical spectroscopy. Comparison of the optical spectra of methemalbumin, ferro-hemalbumin in the absence and presence of 2-methylimidazole, and their carbon monoxide derivatives with horseradish peroxidase (HRP) and its corresponding derivatives indicates that histidine is not present in the first coordination sphere of heme in methemalbumin and that the protein is devoid of a well-defined heme cavity. The complex exhibits peroxidase activity by catalyzing oxidation of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) by hydrogen peroxide. Its activity ( K(M)=433 microM, molar catalytic activity=0.33 s(-1)), however, is considerably lower compared to HRP, indicating differences in the heme environments. Fluorescence intensity decays of Trp214 in HSA and methemalbumin, best fitted to a three-exponential model, gave the lifetimes 7.03 ns (30%), 3.17 ns (38%), and 0.68 ns (32%) for HSA and 8.04 ns (1.7%), 2.42 ns (19.7%), and 0.64 ns (78.6%) for methemalbumin. These lifetime values were further confirmed by a model-independent maximum entropy method. Similarity in the lifetimes and variations in the amplitudes suggest that while conformational heterogeneity of HSA is unperturbed on heme binding, redistribution of the populations of the three conformations occurs and the sub-state associated with the shortest lifetime dominates the total population by approximately 80%. Decay associated spectra (DAS) indicate that the observed lifetime variation with wavelength is predominantly due to ground state heterogeneity, though solvent dipolar relaxation also contributes. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information on motion within the protein together with the whole protein molecule. The binding of heme did not affect the rotational correlation time of the albumin molecule (approximately 20 ns). However, the motion of tryptophan within the protein matrix increased by a factor of approximately 3 (0.46 ns to 0.15 ns). This indicates that while the overall hydrodynamic volume of the albumin molecule remained the same, tryptophan underwent a more rapid internal rotation because of the efficient energy transfer to the bound heme. Optical studies, analysis of lifetime measurements, DAS, and anisotropy measurements together suggest that heme binds to a surface residue. The rapid internal motion of Trp214 during its excited state lifetime for the approximately 80% populated conformer of methemalbumin allows the orientation factor, kappa(2), to approach the average value of 2/3. From the time-resolved fluorescence measurements and the energy transfer calculations on methemalbumin, a Trp214-heme distance of 22 A was deduced.