Pressure, motion, and conformational entropy in molecular recognition by proteins.

The thermodynamics of molecular recognition by proteins is a central determinant of complex biochemistry. For over a half-century, detailed cryogenic structures have provided deep insight into the energetic contributions to ligand binding by proteins. More recently, a dynamical proxy based on NMR-relaxation methods has revealed an unexpected richness in the contributions of conformational entropy to the thermodynamics of ligand binding. Here, we report the pressure dependence of fast internal motion within the ribonuclease barnase and its complex with the protein barstar. In what we believe is a first example, we find that protein dynamics are conserved along the pressure-binding thermodynamic cycle. The femtomolar affinity of the barnase-barstar complex exists despite a penalty by -TΔSconf of +11.7 kJ/mol at ambient pressure. At high pressure, however, the overall change in side-chain dynamics is zero, and binding occurs with no conformational entropy penalty, suggesting an important role of conformational dynamics in the adaptation of protein function to extreme environments. Distinctive clustering of the pressure sensitivity is observed in response to both pressure and binding, indicating the presence of conformational heterogeneity involving less efficiently packed alternative conformation(s). The structural segregation of dynamics observed in barnase is striking and shows how changes in both the magnitude and the sign of regional contributions of conformational entropy to the thermodynamics of protein function are possible.

Protein conformational entropy is not slaved to water.

Conformational entropy can be an important element of the thermodynamics of protein functions such as the binding of ligands. The observed role for conformational entropy in modulating molecular recognition by proteins is in opposition to an often-invoked theory for the interaction of protein molecules with solvent water. The "solvent slaving" model predicts that protein motion is strongly coupled to various aspects of water such as bulk solvent viscosity and local hydration shell dynamics. Changes in conformational entropy are manifested in alterations of fast internal side chain motion that is detectable by NMR relaxation. We show here that the fast-internal side chain dynamics of several proteins are unaffected by changes to the hydration layer and bulk water. These observations indicate that the participation of conformational entropy in protein function is not dictated by the interaction of protein molecules and solvent water under the range of conditions normally encountered.

Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation.

Recent studies suggest that the fast timescale motion of methyl-bearing side chains may play an important role in mediating protein activity. These motions have been shown to encapsulate the residual conformational entropy of the folded state that can potentially contribute to the energetics of protein function. Here, we provide an overview of how to characterize these motions using nuclear magnetic resonance (NMR) spin relaxation methods. The strengths and limitations of several techniques are highlighted in order to assist with experimental design. Particular emphasis is placed on the practical aspects of sample preparation, data collection, data fitting, and statistical analysis. Additionally, discussion of the recently refined "entropy meter" is presented and its use in converting NMR observables to conformational entropy is illustrated. Taken together, these methods should yield new insights into the complex interplay between structure and dynamics in protein function.

Reverse Micelle Encapsulation of Proteins for NMR Spectroscopy.

Reverse micelle (RM) encapsulation of proteins for NMR spectroscopy has many advantages over standard NMR methods such as enhanced tumbling and improved sensitivity. It has opened many otherwise difficult lines of investigation including the study of membrane-associated proteins, large soluble proteins, unstable protein states, and the study of protein surface hydration dynamics. Recent technological developments have extended the ability of RM encapsulation with high structural fidelity for nearly all proteins and thereby allow high-quality state-of-the-art NMR spectroscopy. Optimal conditions are achieved using a streamlined screening protocol, which is described here. Commonly studied proteins spanning a range of molecular weights are used as examples. Very low-viscosity alkane solvents, such as propane or ethane, are useful for studying very large proteins but require the use of specialized equipment to permit preparation and maintenance of well-behaved solutions under elevated pressure. The procedures for the preparation and use of solutions of RMs in liquefied ethane and propane are described. The focus of this chapter is to provide procedures to optimally encapsulate proteins in reverse micelles for modern NMR applications.

Characterizing Protein Hydration Dynamics Using Solution NMR Spectroscopy.

Protein hydration is a critical aspect of protein stability, folding, and function and yet remains difficult to characterize experimentally. Solution NMR offers a route to a site-resolved view of the dynamics of protein-water interactions through the nuclear Overhauser effects between hydration water and the protein in the laboratory (NOE) and rotating (ROE) frames of reference. However, several artifacts and limitations including contaminating contributions from bulk water potentially plague this general approach and the corruption of measured NOEs and ROEs by hydrogen exchange-relayed magnetization. Fortunately, encapsulation of single protein molecules within the water core of a reverse micelle overcomes these limitations. The main advantages are the suppression hydrogen exchange and elimination of bulk water. Here we detail guidelines for the preparation solutions of encapsulated proteins that are suitable for characterization by NOE and ROE spectroscopy. Emphasis is placed on understanding the contribution of detected NOE intensity arising from magnetization relayed by hydrogen exchange. Various aspects of fitting obtained NOE, selectively decoupled NOE, and ROE time courses are illustrated.

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.

Photochemical tyrosine oxidation in the structurally well-defined α3Y protein: proton-coupled electron transfer and a long-lived tyrosine radical.

Tyrosine oxidation-reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The α3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of α3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in α3Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2'-bipyridine)3](3+) oxidant provides high-quality Y32-O• absorption spectra. The rate constant of Y32 oxidation (kPCET) is pH dependent: 1.4 × 10(4) M(-1) s(-1) (pH 5.5), 1.8 × 10(5) M(-1) s(-1) (pH 8.5), 5.4 × 10(3) M(-1) s(-1) (pD 5.5), and 4.0 × 10(4) M(-1) s(-1) (pD 8.5). k(H)/k(D) of Y32 oxidation is 2.5 ± 0.5 and 4.5 ± 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32-O• is 28-58% versus the concentration of [Ru(2,2'-bipyridine)3](3+). Y32-O• decays slowly, t1/2 in the range of 2-10 s, at both pH 5.5 and 8.5, via radical-radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32-O• is discussed relative to the structural properties of the Y32 site. Finally, the static α3Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32-O• come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of α3Y.

Dynamic and thermodynamic response of the Ras protein Cdc42Hs upon association with the effector domain of PAK3.

Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs-PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately -10kcalmol(-1) at 298K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.

High-resolution NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids.

High-resolution multi-dimensional solution NMR is unique as a biophysical and biochemical tool in its ability to examine both the structure and dynamics of macromolecules at atomic resolution. Conventional solution NMR approaches, however, are largely limited to examinations of relatively small (<25kDa) molecules, mostly due to the spectroscopic consequences of slow rotational diffusion. Encapsulation of macromolecules within the protective nanoscale aqueous interior of reverse micelles dissolved in low viscosity fluids has been developed as a means through which the 'slow tumbling problem' can be overcome. This approach has been successfully applied to diverse proteins and nucleic acids ranging up to 100kDa, considerably widening the range of biological macromolecules to which conventional solution NMR methodologies may be applied. Recent advances in methodology have significantly broadened the utility of this approach in structural biology and molecular biophysics.

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.

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 13C labeling strategy reveals a range of aromatic side chain motion in calmodulin.

NMR relaxation experiments often require site-specific isotopic enrichment schemes in order to allow for quantitative interpretation. Here we describe a new labeling scheme for site-specific (13)C-(1)H enrichment of a single ortho position of aromatic amino acid side chains in an otherwise perdeuterated background by employing a combination of [4-(13)C]erythrose and deuterated pyruvate during growth on deuterium oxide. This labeling scheme largely eliminates undesired contributions to (13)C relaxation and greatly simplifies the fitting of relaxation data using the Lipari-Szabo model-free formalism. This approach is illustrated with calcium-saturated vertebrate calmodulin and oxidized flavodoxin from Cyanobacterium anabaena . Analysis of (13)C relaxation in the aromatic groups of calcium-saturated calmodulin indicates a wide range of motion in the subnanosecond time regime.

Reversible phenol oxidation and reduction in the structurally well-defined 2-Mercaptophenol-α3C protein.

2-Mercaptophenol-α3C serves as a biomimetic model for enzymes that use tyrosine residues in redox catalysis and multistep electron transfer. This model protein was tailored for electrochemical studies of phenol oxidation and reduction with specific emphasis on the redox-driven protonic reactions occurring at the phenol oxygen. This protein contains a covalently modified 2-mercaptophenol-cysteine residue. The radical site and the phenol compound were specifically chosen to bury the phenol OH group inside the protein. A solution nuclear magnetic resonance structural analysis (i) demonstrates that the synthetic 2-mercaptophenol-α3C model protein behaves structurally as a natural protein, (ii) confirms the design of the radical site, (iii) reveals that the ligated phenol forms an interhelical hydrogen bond to glutamate 13 (phenol oxygen-carboxyl oxygen distance of 3.2 ± 0.5 Å), and (iv) suggests a proton-transfer pathway from the buried phenol OH (average solvent accessible surface area of 3 ± 5%) via glutamate 13 (average solvent accessible surface area of the carboxyl oxygens of 37 ± 18%) to the bulk solvent. A square-wave voltammetry analysis of 2-mercaptophenol-α3C further demonstrates that (v) the phenol oxidation-reduction cycle is reversible, (vi) formal phenol reduction potentials can be obtained, and (vii) the phenol-O(•) state is long-lived with an estimated lifetime of ≥180 millisecond. These properties make 2-mercaptophenol-α3C a unique system for characterizing phenol-based proton-coupled electron transfer in a low-dielectric and structured protein environment.

The dynamical response of hen egg white lysozyme to the binding of a carbohydrate ligand.

It has become clear that the binding of small and large ligands to proteins can invoke significant changes in side chain and main chain motion in the fast picosecond to nanosecond timescale. Recently, the use of a "dynamical proxy" has indicated that changes in these motions often reflect significant changes in conformational entropy. These entropic contributions are sometimes of the same order as the total entropy of binding. Thus, it is important to understand the connections amongst motion between the manifold of states accessible to the native state of proteins, the corresponding entropy, and how this impacts the overall energetics of protein function. The interaction of proteins with carbohydrate ligands is central to a range of biological functions. Here, we examine a classic carbohydrate interaction with an enzyme: the binding of wild-type hen egg white lysozyme (HEWL) to the natural, competitive inhibitor chitotriose. Using NMR relaxation experiments, backbone amide and side chain methyl axial order parameters were obtained across apo and chitotriose-bound HEWL. Upon binding, changes in the apparent amplitude of picosecond to nanosecond main chain and side chain motions are seen across the protein. Indeed, binding of chitotriose renders a large contiguous fraction of HEWL effectively completely rigid. Changes in methyl flexibility are most pronounced closest to the binding site, but average to only a small overall change in the dynamics across the protein. The corresponding change in conformational entropy is unfavorable and estimated to be a significant fraction of the total binding entropy.

Solution structure of the core SMN-Gemin2 complex.

In humans, assembly of spliceosomal snRNPs (small nuclear ribonucleoproteins) begins in the cytoplasm where the multi-protein SMN (survival of motor neuron) complex mediates the formation of a seven-membered ring of Sm proteins on to a conserved site of the snRNA (small nuclear RNA). The SMN complex contains the SMN protein Gemin2 and several additional Gemins that participate in snRNP biosynthesis. SMN was first identified as the product of a gene found to be deleted or mutated in patients with the neurodegenerative disease SMA (spinal muscular atrophy), the leading genetic cause of infant mortality. In the present study, we report the solution structure of Gemin2 bound to the Gemin2-binding domain of SMN determined by NMR spectroscopy. This complex reveals the structure of Gemin2, how Gemin2 binds to SMN and the roles of conserved SMN residues near the binding interface. Surprisingly, several conserved SMN residues, including the sites of two SMA patient mutations, are not required for binding to Gemin2. Instead, they form a conserved SMN/Gemin2 surface that may be functionally important for snRNP assembly. The SMN-Gemin2 structure explains how Gemin2 is stabilized by SMN and establishes a framework for structure-function studies to investigate snRNP biogenesis as well as biological processes involving Gemin2 that do not involve snRNP assembly.

Electrochemical and structural properties of a protein system designed to generate tyrosine Pourbaix diagrams.

This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue α(3)Y three-helix bundle. α(3)Y contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from α(3)Y at both acidic (pH 5.6) and alkaline (pH 8.3) conditions. The observed Faradaic response is uniquely associated with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton-coupled electron-transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold. α(3)Y was re-engineered with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring. α(3)Y-K29H and α(3)Y-K36H each contain a histidine residue whose protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical content, tertiary structure, solution aggregation state, and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3-4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects.

Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids.

Comprehensive application of solution NMR spectroscopy to studies of macromolecules remains fundamentally limited by the molecular rotational correlation time. For proteins, molecules larger than 30 kDa require complex experimental methods, such as TROSY in conjunction with isotopic labeling schemes that are often expensive and generally reduce the potential information available. We have developed the reverse micelle encapsulation strategy as an alternative approach. Encapsulation of proteins within the protective nano-scale water pool of a reverse micelle dissolved in ultra-low viscosity nonpolar solvents overcomes the slow tumbling problem presented by large proteins. Here, we characterize the contributions from the various components of the protein-containing reverse micelle system to the rotational correlation time of the encapsulated protein. Importantly, we demonstrate that the protein encapsulated in the reverse micelle maintains a hydration shell comparable in size to that seen in bulk solution. Using moderate pressures, encapsulation in ultra-low viscosity propane or ethane can be used to magnify this advantage. We show that encapsulation in liquid ethane can be used to reduce the tumbling time of the 43 kDa maltose binding protein from ~23 to ~10 ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 43 kDa encapsulated maltose binding protein dissolved in liquid ethane, which is typically impossible for proteins of such size without use of extensive deuteration or the TROSY effect.

NMR analysis of the alphaIIb beta3 cytoplasmic interaction suggests a mechanism for integrin regulation.

The integrin αIIbß3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting αIIbß3, the TM and cytoplasmic domains of αIIb and ß3 form a heterodimer that constrains αIIbß3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogen-deuterium exchange of this complex in micelles showed that the αIIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the ß3 cytoplasmic domain. The ß3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the ß3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the ß3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites.

The role of conformational entropy in molecular recognition by calmodulin.

The physical basis for high-affinity interactions involving proteins is complex and potentially involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed from molecular structures. We have recently used changes in conformational dynamics as a proxy for changes in conformational entropy of calmodulin upon association with domains from regulated proteins. The apparent change in conformational entropy was linearly related to the overall binding entropy. This view warrants a more quantitative foundation. Here we calibrate an 'entropy meter' using an experimental dynamical proxy based on NMR relaxation and show that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. Furthermore, the distribution of motion at the interface between the target domain and calmodulin is surprisingly noncomplementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.