A fine balance of hydrophobic-electrostatic communication pathways in a pH-switching protein.

Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.

Using biochemistry and biophysics to extinguish androgen receptor signaling in prostate cancer.

Castration resistant prostate cancer (CRPC) continues to be androgen receptor (AR) driven. Inhibition of AR signaling in CRPC could be advanced using state-of-the-art biophysical and biochemical techniques. Structural characterization of AR and its complexes by cryo-electron microscopy would advance the development of N-terminal domain (NTD) and ligand-binding domain (LBD) antagonists. The structural basis of AR function is unlikely to be determined by any single structure due to the intrinsic disorder of its NTD, which not only interacts with coregulators but likely accounts for the constitutive activity of AR-splice variants (SV), which lack the LBD and emerge in CRPC. Using different AR constructs lacking the LBD, their effects on protein folding, DNA binding, and transcriptional activity could reveal how interdomain coupling explains the activity of AR-SVs. The AR also interacts with coregulators that promote chromatin looping. Elucidating the mechanisms involved can identify vulnerabilities to treat CRPC, which do not involve targeting the AR. Phosphorylation of the AR coactivator MED-1 by CDK7 is one mechanism that can be blocked by the use of CDK7 inhibitors. CRPC gains resistance to AR signaling inhibitors (ARSI). Drug resistance may involve AR-SVs, but their role requires their reliable quantification by SILAC-mass spectrometry during disease progression. ARSI drug resistance also occurs by intratumoral androgen biosynthesis catalyzed by AKR1C3 (type 5 17ß-hydroxysteroid dehydrogenase), which is unique in that its acts as a coactivator of AR. Novel bifunctional inhibitors that competitively inhibit AKR1C3 and block its coactivator function could be developed using reverse-micelle NMR and fragment-based drug discovery.

Dynamic activation and regulation of the mitogen-activated protein kinase p38.

Mitogen-activated protein kinases, which include p38, are essential for cell differentiation and autophagy. The current model for p38 activation involves activation-loop phosphorylation with subsequent substrate binding leading to substrate phosphorylation. Despite extensive efforts, the molecular mechanism of activation remains unclear. Here, using NMR spectroscopy, we show how the modulation of protein dynamics across timescales activates p38. We find that activation-loop phosphorylation does not change the average conformation of p38; rather it quenches the loop ps-ns dynamics. We then show that substrate binding to nonphosphorylated and phosphorylated p38 results in uniform µs-ms backbone dynamics at catalytically essential regions and across the entire molecule, respectively. Together, these results show that phosphorylation and substrate binding flatten the energy landscape of the protein, making essential elements of allostery and activation dynamically accessible. The high degree of structural conservation among ser/thr kinases suggests that elements of this mechanism may be conserved across the kinase family.

Entropy in molecular recognition by proteins.

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein-ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein-ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein-ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or "entropy meter" also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water-protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins.

Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy.

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side-chain dynamics of the α-helical sensory rhodopsin II and the ß-barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl-bearing side-chain motion that is largely independent of membrane mimetic. The methyl-bearing side chains of both proteins are, on average, more dynamic in the ps-ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane-protein functions, including ligand binding, allostery, and signaling.

Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology.

Pressure and temperature are the two fundamental variables of thermodynamics. Temperature and chemical perturbation are central experimental tools for the exploration of macromolecular structure and dynamics. Though it has long been recognized that hydrostatic pressure offers a complementary and often unique view of macromolecular structure, stability and dynamics, it has not been employed nearly as much. For solution NMR applications the limited use of high-pressure is undoubtedly traced to difficulties of employing pressure in the context of modern multinuclear and multidimensional NMR. Limitations in pressure tolerant NMR sample cells have been overcome and enable detailed studies of macromolecular energy landscapes, hydration, dynamics and function. Here we review the practical considerations for studies of biological macromolecules at elevated pressure, with a particular emphasis on applications in protein biophysics and structural biology.

Improving yields of deuterated, methyl labeled protein by growing in H2O.

Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the 15N-1H TROSY and 13C-1H3 methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E. coli) during growth on media prepared in D2O leads to many difficulties and limitations, such as cell toxicity, decreased yield, and the need to unfold or destabilize proteins for back exchange of amide protons. These issues are exacerbated for non-ideal systems such as membrane proteins. Expression of protein during growth in H2O, with the addition of 2H-labeled amino acids derived from algal extract, can potentially avoid these issues. We demonstrate a novel fermentation methodology for high-density bacterial growth in H2O M9 medium that allows for appropriate isotopic labeling and deuteration. Yields are significantly higher than those achieved in D2O M9 for a variety of protein targets while still achieving 75-80% deuteration. Because the procedure does not require bulk D2O or deuterated glucose, the cost per liter of growth medium is significantly decreased; taking into account improvements in yield, these savings can be quite dramatic. Triple-labeled protein is also efficiently produced including specific 13CH3 labeling of isoleucine, leucine, and valine using the traditional ILV precursors in combination with an ILV-depleted mix of 2H/15N amino acids. These results are demonstrated for the membrane protein sensory rhodopsin II and the soluble proteins human aldoketoreductase AKR1c3, human ubiquitin, and bacterial flavodoxin. Limitations of the approach in the context of very large molecular weight proteins are illustrated using the bacterial Lac repressor transcription factor.

Role of cavities and hydration in the pressure unfolding of T4 lysozyme.

It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.

Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN.

The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (bb ∼ 0.23 Å) using NMR-based methods and is closely similar to the cryogenic crystal structure (bb ∼ 1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.

Accurate determination of rates from non-uniformly sampled relaxation data.

The application of non-uniform sampling (NUS) to relaxation experiments traditionally used to characterize the fast internal motion of proteins is quantitatively examined. Experimentally acquired Poisson-gap sampled data reconstructed with iterative soft thresholding are compared to regular sequentially sampled (RSS) data. Using ubiquitin as a model system, it is shown that 25 % sampling is sufficient for the determination of quantitatively accurate relaxation rates. When the sampling density is fixed at 25 %, the accuracy of rates is shown to increase sharply with the total number of sampled points until eventually converging near the inherent reproducibility of the experiment. Perhaps contrary to some expectations, it is found that accurate peak height reconstruction is not required for the determination of accurate rates. Instead, inaccuracies in rates arise from inconsistencies in reconstruction across the relaxation series that primarily manifest as a non-linearity in the recovered peak height. This indicates that the performance of an NUS relaxation experiment cannot be predicted from comparison of peak heights using a single RSS reference spectrum. The generality of these findings was assessed using three alternative reconstruction algorithms, eight different relaxation measurements, and three additional proteins that exhibit varying degrees of spectral complexity. From these data, it is revealed that non-linearity in peak height reconstruction across the relaxation series is strongly correlated with errors in NUS-derived relaxation rates. Importantly, it is shown that this correlation can be exploited to reliably predict the performance of an NUS-relaxation experiment by using three or more RSS reference planes from the relaxation series. The RSS reference time points can also serve to provide estimates of the uncertainty of the sampled intensity, which for a typical relaxation times series incurs no penalty in total acquisition time.

Characterization of Cetyltrimethylammonium Bromide/Hexanol Reverse Micelles by Experimentally Benchmarked Molecular Dynamics Simulations.

Encapsulation of small molecules, proteins, and other macromolecules within the protective water core of reverse micelles is emerging as a powerful strategy for a variety of applications. The cationic surfactant cetyltrimethylammonium bromide (CTAB) in combination with hexanol as a cosurfactant is particularly useful in the context of solution NMR spectroscopy of encapsulated proteins. Small-angle X-ray and neutron scattering is employed to investigate the internal structure of the CTAB/hexanol reverse micelle particle under conditions appropriate for high-resolution NMR spectroscopy. The scattering profiles are used to benchmark extensive molecular dynamics simulations of this reverse micelle system and indicate that the parameters used in these simulations recapitulate experimental results. Scattering profiles and simulations indicate formation of homogeneous solutions of small approximately spherical reverse micelle particles at a water loading of 20 composed of ∼150 CTAB and 240 hexanol molecules. The 3000 waters comprising the reverse micelle core show a gradient of translational diffusion that reaches that of bulk water at the center. Rotational diffusion is slowed relative to bulk throughout the water core, with the greatest slowing near the CTAB headgroups. The 5 Šthick interfacial region of the micelle consists of overlapping layers of Br(-) enriched water, CTAB headgroups, and hexanol hydroxyl groups, containing about one-third of the total water. This study employs well-parametrized MD simulations, X-ray and neutron scattering, and electrostatic theory to illuminate fundamental properties of CTAB/hexanol reverse micelle size, shape, partitioning, and water behavior.

Optimized expression and purification of biophysical quantities of Lac repressor and Lac repressor regulatory domain.

The recombinant production of Lac repressor (LacI) in Escherichia coli is complicated by its ubiquitous use as a regulatory element in commercially-available expression vectors and host strains. While LacI-regulated expression systems are often used to produce recombinant LacI, the product can be heterogeneous and unsuitable for some studies. Alternative approaches include using unregulated vectors which typically suffer from low yield or vectors with promoters induced by metabolically active sugars which can dilute isotope labels necessary for certain biophysical studies. Here, an optimized expression system and isolation protocol for producing various constructs of LacI is introduced which eliminates these complications. The expression vector is an adaptation of the pASK backbone wherein expression of the lacI gene is regulated by an anhydrotetracyline inducible tetA promoter and the host strain lacks the lacI gene. Typical yields in highly deuterated minimal medium are nearly 2-fold greater than those previously reported. Notably, the new expression system is also able to produce the isolated regulatory domain of LacI without co-expression of the full-length protein and without any defects in cell viability, eliminating the inconvenient requirement for strict monitoring of cell densities during pre-culturing. Typical yields in highly deuterated minimal medium are significantly greater than those previously reported. Characterization by solution NMR shows that LacI constructs produced using this expression system are highly homogenous and functionally active.

On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.

Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the conformational entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements.

Elementary tetrahelical protein design for diverse oxidoreductase functions.

Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications.

A sharp thermal transition of fast aromatic-ring dynamics in ubiquitin.

Aromatic amino acid side chains have a rich role within proteins and are often central to their structure and function. Suitable isotopic-labelling strategies enable studies of sub-nanosecond aromatic-ring dynamics using solution NMR relaxation methods. Surprisingly, it was found that the three aromatic side chains in human ubiquitin show a sharp thermal dynamical transition at approximately 312 K. Hydrostatic pressure has little effect on the low-temperature behavior, but somewhat decreases the amplitude of motion in the high-temperature regime. Therefore, below the transition temperature, ring motion is largely librational. Above this temperature, a complete ring-rotation process that is fully consistent with a continuous diffusion not requiring the transient creation of a large activated free volume occurs. Molecular dynamics simulations qualitatively corroborate this view and reinforce the notion that the dynamical character of the protein interior has much more liquid-alkane-like properties than previously appreciated.

Banding of NMR-derived methyl order parameters: implications for protein dynamics.

Our understanding of protein folding, stability, and function has begun to more explicitly incorporate dynamical aspects. Nuclear magnetic resonance has emerged as a powerful experimental method for obtaining comprehensive site-resolved insight into protein motion. It has been observed that methyl-group motion tends to cluster into three "classes" when expressed in terms of the popular Lipari-Szabo model-free squared generalized order parameter. Here the origins of the three classes or bands in the distribution of order parameters are examined. As a first step, a Bayesian based approach, which makes no a priori assumption about the existence or number of bands, is developed to detect the banding of Oaxis2 values derived either from NMR experiments or molecular dynamics simulations. The analysis is applied to seven proteins with extensive molecular dynamics simulations of these proteins in explicit water to examine the relationship between O2 and fine details of the motion of methyl bearing side chains. All of the proteins studied display banding, with some subtle differences. We propose a very simple yet plausible physical mechanism for banding. Finally, our Bayesian method is used to analyze the measured distributions of methyl group motions in the catabolite activating protein and several of its mutants in various liganded states and discuss the functional implications of the observed banding to protein dynamics and function.

Optimized reverse micelle surfactant system for high-resolution NMR spectroscopy of encapsulated proteins and nucleic acids dissolved in low viscosity fluids.

An optimized reverse micelle surfactant system has been developed for solution nuclear magnetic resonance studies of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. Comprising the nonionic 1-decanoyl-rac-glycerol and the zwitterionic lauryldimethylamine-N-oxide (10MAG/LDAO), this mixture is shown to efficiently encapsulate a diverse set of proteins and nucleic acids. Chemical shift analyses of these systems show that high structural fidelity is achieved upon encapsulation. The 10MAG/LDAO surfactant system reduces the molecular reorientation time for encapsulated macromolecules larger than ~20 kDa leading to improved overall NMR performance. The 10MAG/LDAO system can also be used for solution NMR studies of lipid-modified proteins. New and efficient strategies for optimization of encapsulation conditions are described. 10MAG/LDAO performs well in both the low viscosity pentane and ultralow viscosity liquid ethane and therefore will serve as a general surfactant system for initiating solution NMR studies of proteins and nucleic acids.

Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins.

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ∼-93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules.