The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins.

RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

An analysis of double-quantum coherence ESR in an N-spin system: Analytical expressions and predictions.

Electron spin resonance pulsed dipolar spectroscopy (PDS) has become popular in protein 3D structure analysis. PDS studies yield distance distributions between a pair or multiple pairs of spin probes attached to protein molecules, which can be used directly in structural studies or as constraints in theoretical predictions. Double-quantum coherence (DQC) is a highly sensitive and accurate PDS technique to study protein structures in the solid state and under physiologically relevant conditions. In this work, we have derived analytical expressions for the DQC signal for a system with N-dipolar coupled spin-1/2 particles in the solid state. The expressions are integrated over the relevant spatial parameters to obtain closed form DQC signal expressions. These expressions contain the concentration-dependent "instantaneous diffusion" and the background signal. For micromolar and lower concentrations, these effects are negligible. An approximate analysis is provided for cases of finite pulses. The expressions obtained in this work should improve the analysis of DQC experimental data significantly, and the analytical approach could be extended easily to a wide range of magnetic resonance phenomena.

Thermal Degradation of Thaumatin at Low pH and Its Prevention Using Alkyl Gallates.

Thaumatin, a potent sweet tasting protein extracted from the Katemfe Plant, is emerging as a natural alternative to synthetic non-nutritive sweeteners and flavor enhancer. As a food additive, its stability within the food matrix during thermal processing is of great interest to the food industry. When heated under neutral or basic conditions, thaumatin was found to lose its sweetness due to protein aggregation caused by sulfhydryl catalyzed disulfide bond interchange. At lower pH, while thaumatin was also found to lose sweetness after heating, it does so at a slower rate and shows more resistance to sweetness loss. SDS-PAGE indicated that thaumatin fragmented into multiple smaller pieces under heating in acidic pH. Using BEMPO-3, a lipophilic spin trap, we were able to detect the presence of a free-radical within the hydrophobic region of the protein during heating. Protein carbonyl content, a byproduct of protein oxidation, also increased upon heating, providing additional evidence for protein cleavage by a radical pathway. Hexyl gallate successfully inhibited the radical generation as well as protein carbonyl formation of thaumatin during heating.

Negatively charged residues in the membrane ordering activity of SARS-CoV-1 and -2 fusion peptides.

Entry of coronaviruses into host cells is mediated by the viral spike protein. Previously, we identified the bona fide fusion peptides (FPs) for severe acute respiratory syndrome coronavirus ("SARS-1") and severe acute respiratory syndrome coronavirus-2 ("SARS-2") using electron spin resonance spectroscopy. We also found that their FPs induce membrane ordering in a Ca2+-dependent fashion. Here we study which negatively charged residues in SARS-1 FP are involved in this binding, to build a topological model and clarify the role of Ca2+. Our systematic mutation study on the SARS-1 FP shows that all six negatively charged residues contribute to the FP's membrane ordering activity, with D812 the dominant residue. The corresponding SARS-2 residue D830 plays an equivalent role. We provide a topological model of how the FP binds Ca2+ ions: its two segments FP1 and FP2 each bind one Ca2+. The binding of Ca2+, the folding of FP (both studied by isothermal titration calorimetry experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca2+ helps the folding of FP in membranes to enhance the ordering activity. Using a novel pseudotyped viral particle-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole spike protein in its trimer form in real time. We found that the SARS-1 and SARS-2 pseudotyped viral particles also induce membrane ordering to the extent that separate FPs do, and mutations of the negatively charged residues also significantly suppress the membrane ordering activity. However, the slower kinetics of the FP ordering activity versus that of the pseudotyped viral particle suggest the need for initial trimerization of the FPs.

Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346.

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light-sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

Theory and Least Squares Fitting of CW ESR Saturation Spectra Using the MOMD Model.

CW saturation experiments are widely used in ESR studies of relaxation processes in proteins and lipids. We develop the theory of saturation in ESR spectra in terms of its close relation with that of 2D-ELDOR. Our treatment of saturation is then based on the microscopic order macroscopic disorder (MOMD) model and can be used to fit the full CW saturation spectrum, rather than fitting just the peak-peak amplitude as a function of microwave field B 1 as is commonly done. This requires fewer experiments to yield effects on T 1, as well as provides a more extensive dynamic structural picture, for example, for scanning experiments on different protein sites. The code is released as a publicly available software package in Python that can be used to fit CW saturation spectra from biological samples of interest.

Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe-4S] to a [4Fe-4S] Cluster in Dph1-Dph2.

All radical S-adenosylmethionine (radical-SAM) enzymes, including the noncanonical radical-SAM enzyme diphthamide biosynthetic enzyme Dph1-Dph2, require at least one [4Fe-4S](Cys)3 cluster for activity. It is well-known in the radical-SAM enzyme community that the [4Fe-4S](Cys)3 cluster is extremely air-sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1-Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe-4S] cluster is easily degraded into a [3Fe-4S] cluster. Remarkably, the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe-4S] cluster in Dph1-Dph2 to a functional [4Fe-4S] cluster during the radical-SAM enzyme catalytic cycle. This mechanism to maintain radical-SAM enzyme activity in aerobic environments is likely general, and Dph3-like proteins may exist to keep other radical-SAM enzymes functional in aerobic environments.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity.

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.

Extraction of Weak Spectroscopic Signals with High Fidelity: Examples from ESR.

Noise impedes experimental studies by reducing signal resolution and/or suppressing weak signals. Signal averaging and filtering are the primary methods used to reduce noise, but they have limited effectiveness and lack capabilities to recover signals at low signal-to-noise ratios (SNRs). We utilize a wavelet transform-based approach to effectively remove noise from spectroscopic data. The wavelet denoising method we use is a significant improvement on standard wavelet denoising approaches. We demonstrate its power in extracting signals from noisy spectra on a variety of signal types ranging from hyperfine lines to overlapped peaks to weak peaks overlaid on strong ones, drawn from electron-spin-resonance spectroscopy. The results show that one can accurately extract details of complex spectra, including retrieval of very weak ones. It accurately recovers signals at an SNR of ∼1 and improves the SNR by about 3 orders of magnitude with high fidelity. Our examples show that one is now able to address weaker SNR signals much better than by previous methods. This new wavelet approach can be successfully applied to other spectroscopic signals.

Transport domain unlocking sets the uptake rate of an aspartate transporter.

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

Microsecond dynamics in proteins by two-dimensional ESR. II. Addressing computational challenges.

Two-dimensional electron-electron double resonance (2D-ELDOR) provides extensive insight into molecular motions. Recent developments permitting experiments at higher frequencies (95 GHz) provide molecular orientational resolution, enabling a clearer description of the nature of the motions. In previous work, we provided simulations for the case of domain motions within proteins that are themselves slowly tumbling in a solution. In order to perform these simulations, it was found that the standard approach of solving the relevant stochastic Liouville equation using the efficient Lanczos algorithm for this case breaks down, so algorithms were employed that rely on the Arnoldi iteration. While they lead to accurate simulations, they are very time-consuming. In this work, we focus on a variant known as the rational Arnoldi algorithm. We show that this can achieve a significant reduction in computation time. The stochastic Liouville matrix, which is of very large dimension, N, is first reduced to a much smaller dimension, m, e.g., from N ∼ O(104) to m ∼ 60, that spans the relevant Krylov subspace from which the spectrum is predicted. This requires the selection of the m frequency shifts to be utilized. A method of adaptive shift choice is introduced to optimize this selection. We also find that these procedures help in optimizing the pruning procedure that greatly reduces the dimension of the initial N dimensional stochastic Liouville matrix in such subsequent computations.

Cofactors are essential constituents of stable and seeding-active tau fibrils.

Amyloid fibrils are cross-ß-rich aggregates that are exceptionally stable forms of protein assembly. Accumulation of tau amyloid fibrils is involved in many neurodegenerative diseases, including Alzheimer's disease (AD). Heparin-induced aggregates have been widely used and assumed to be a good tau amyloid fibril model for most biophysical studies. Here we show that mature fibrils made of 4R tau variants, prepared with heparin or RNA, spontaneously depolymerize and release monomers when their cofactors are removed. We demonstrate that the cross-ß-sheet assembly formed in vitro with polyanion addition is unstable at room temperature. We furthermore demonstrate high seeding capacity with transgenic AD mouse brain-extracted tau fibrils in vitro that, however, is exhausted after one generation, while supplementation with RNA cofactors resulted in sustained seeding over multiple generations. We suggest that tau fibrils formed in brains are supported by unknown cofactors and inhere higher-quality packing, as reflected in a more distinct conformational arrangement in the mouse fibril-seeded, compared with heparin-induced, tau fibrils. Our study suggests that the role of cofactors in tauopathies is a worthy focus of future studies, as they may be viable targets for diagnosis and therapeutics.

Microsecond Exchange Processes Studied by Two-Dimensional ESR at 95 GHz.

Exchange processes which include conformational change, protonation/deprotonation, and binding equilibria are routinely studied by 2D exchange NMR techniques, where information about the exchange of nuclei between environments with different NMR shifts is obtained from the development of cross-peaks. Whereas 2D NMR enables the real time study of millisecond and slower exchange processes, 2D ESR in the form of 2D-ELDOR (two-dimensional electron-electron double resonance) has the potential for such studies over the nanosecond to microsecond real time scales. Cross-peak development due to chemical exchange has been seen previously for semiquinones in ESR, but this is not possible for most common ESR probes, such as nitroxides, studied at typical ESR frequencies because, unlike NMR, the exchanging states yield ESR signals that are not resolved from each other within their respective line widths. But at 95 GHz, it becomes possible to resolve them in many cases because of the increased g-factor resolution. The 95 GHz instrumental developments occurring at ACERT now enable such studies. We demonstrate these new capabilities in two studies: (A) the protonation/deprotonation process for a pH-sensitive imidazoline spin label in aqueous solution where the exchange rate and the population ratio of the exchanging states are controlled by the concentration and pH of the buffer solution, respectively, and (B) a nitroxide radical partitioning between polar (aqueous) and nonpolar (phospholipid) environments in multilamellar lipid vesicles, where the cross-peak development arises from the exchange of the nitroxide between the two phases. This work represents the first example of the observation and analysis of cross-peaks arising from chemical exchange processes involving nitroxide spin labels.

High-yield production in E. coli and characterization of full-length functional p13II protein from human T-cell leukemia virus type 1.

Human T-cell leukemia virus type 1 is an oncovirus that causes aggressive adult T-cell leukemia but is also responsible for severe neurodegenerative and endocrine disorders. Combatting HTLV-1 infections requires a detailed understanding of the viral mechanisms in the host. Therefore, in vitro studies of important virus-encoded proteins would be critical. Our focus herein is on the HTLV-1-encoded regulatory protein p13II, which interacts with the inner mitochondrial membrane, increasing its permeability to cations (predominantly potassium, K+). Thereby, this protein affects mitochondrial homeostasis. We report on our progress in developing specific protocols for heterologous expression of p13II in E. coli, and methods for its purification and characterization. We succeeded in producing large quantities of highly-pure full-length p13II, deemed to be its fully functional form. Importantly, our particular approach based on the fusion of ubiquitin to the p13II C-terminus was instrumental in increasing the persistently low expression of soluble p13II in its native form. We subsequently developed approaches for protein spin labeling and a conformation study using double electron-electron resonance (DEER) spectroscopy and a fluorescence-based cation uptake assay for p13II in liposomes. Our DEER results point to large protein conformation changes occurring upon transition from the soluble to the membrane-bound state. The functional assay on p13II-assisted transport of thallium (Tl+) through the membrane, wherein Tl+ substituted for K+, suggests transmembrane potential involvement in p13II function. Our study lays the foundation for expansion of in vitro functional and structural investigations on p13II and would aid in the development of structure-based protein inhibitors and markers.

Conformational Dynamics in Extended RGD-Containing Peptides.

RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.

Microsecond dynamics in proteins by two-dimensional ESR: Predictions.

Two-dimensional electron-electron double resonance (2D-ELDOR) provides extensive insight into molecular motions. Recent developments permitting experiments at higher frequencies (95 GHz) provide molecular orientational resolution, enabling a clearer description of the nature of the motions. In this work, simulations are provided for the example of domain motions within proteins that are themselves slowly tumbling in solution. These show the nature of the exchange cross-peaks that are predicted to develop in real time from such domain motions. However, we find that the existing theoretical methods for computing 2D-ELDOR experiments over a wide motional range begin to fail seriously when applied to very slow motions characteristic of proteins in solution. One reason is the failure to obtain accurate eigenvectors and eigenvalues of the complex symmetric stochastic Liouville matrices describing the experiment when computed by the efficient Lanczos algorithm in the range of very slow motion. Another, perhaps more serious, issue is that these matrices are "non-normal," such that for the very slow motional range even rigorous diagonalization algorithms do not yield the correct eigenvalues and eigenvectors. We have employed algorithms that overcome both these issues and lead to valid 2D-ELDOR predictions even for motions approaching the rigid limit. They are utilized to describe the development of cross-peaks in 2D-ELDOR at 95 GHz for a particular case of domain motion.

The asymmetric function of Dph1-Dph2 heterodimer in diphthamide biosynthesis.

Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation elongation factor 2 (EF2). In the first step of diphthamide biosynthesis, a [4Fe-4S] cluster-containing radical SAM enzyme, Dph1-Dph2 heterodimer in eukaryotes or Dph2 homodimer in archaea, cleaves S-adenosylmethionine and transfers the 3-amino-3-carboxypropyl group to EF2. It was demonstrated previously that for the archaeal Dph2 homodimer, only one [4Fe-4S] cluster is necessary for the in vitro activity. Here, we demonstrate that for the eukaryotic Dph1-Dph2 heterodimer, the [4Fe-4S] cluster-binding cysteine residues in each subunit are required for diphthamide biosynthesis to occur in vivo. Furthermore, our in vitro reconstitution experiments with Dph1-Dph2 mutants suggested that the Dph1 cluster serves a catalytic role, while the Dph2 cluster facilitates the reduction of the Dph1 cluster by the physiological reducing system Dph3/Cbr1/NADH. Our results reveal the asymmetric functional roles of the Dph1-Dph2 heterodimer and may help to understand how the Fe-S clusters in radical SAM enzymes are reduced in biology.

Singular Value Decomposition Method To Determine Distance Distributions in Pulsed Dipolar Electron Spin Resonance: II. Estimating Uncertainty.

This paper is a continuation of the method introduced by Srivastava and Freed (2017) that is a new method based on truncated singular value decomposition (TSVD) for obtaining physical results from experimental signals without any need for Tikhonov regularization or other similar methods that require a regularization parameter. We show here how to estimate the uncertainty in the SVD-generated solutions. The uncertainty in the solution may be obtained by finding the minimum and maximum values over which the solution remains converged. These are obtained from the optimum range of singular value contributions, where the width of this region depends on the solution point location (e.g., distance) and the signal-to-noise ratio (SNR) of the signal. The uncertainty levels typically found are very small with substantial SNR of the (denoised) signal, emphasizing the reliability of the method. With poorer SNR, the method is still satisfactory but with greater uncertainty, as expected. Pulsed dipolar electron spin resonance spectroscopy experiments are used as an example, but this TSVD approach is general and thus applicable to any similar experimental method wherein singular matrix inversion is needed to obtain the physically relevant result. We show that the Srivastava-Freed TSVD method along with the estimate of uncertainty can be effectively applied to pulsed dipolar electron spin resonance signals with SNR > 30, and even for a weak signal (e.g., SNR ≈ 3) reliable results are obtained by this method, provided the signal is first denoised using wavelet transforms (WavPDS).

Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy.

An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.