Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy.
ABSTRACT: Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)-ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of (15)N, (1)HN, (13)C(alpha), and (13)CO chemical shifts, along with (1)HN-(15)N, (1)H(alpha)-(13)C(alpha), and (1)HN-(13)CO residual dipolar couplings and (13)CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with (1)HN-(15)N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins.
Project description:Residual dipolar couplings (RDCs) were used as restraints in fully solvated molecular dynamics simulations of reduced substrate- and carbonmonoxy-bound cytochrome P450(cam) (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The (1)D(NH) residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site and largely support previously reported differences between the open and closed states of CYP101A1.
Project description:The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in ∼ 50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of site-specific pK(a)s and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often "invisible" in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine (13)Cγ, (13)Cδ2 chemical shifts and (1)Hε-(13)Cε one-bond scalar couplings. The methodology is first validated and then used to obtain pKa values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pKa values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5-6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.
Project description:The focus of structural biology is on studies of the highly populated, ground states of biological molecules; states that are only sparsely and transiently populated are more difficult to probe because they are invisible to most structural methods. Yet, such states can play critical roles in biochemical processes such as ligand binding, enzyme catalysis, and protein folding. A description of these states in terms of structure and dynamics is, therefore, of great importance. Here, we present a method, based on relaxation dispersion NMR spectroscopy of weakly aligned molecules in a magnetic field, that can provide such a description by direct measurement of backbone amide bond vector orientations in transient, low populated states that are not observable directly. Such information, obtained through the measurement of residual dipolar couplings, has until now been restricted to proteins that produce observable spectra. The methodology is applied and validated in a study of the binding of a target peptide to an SH3 domain from the yeast protein Abp1p and subsequently used in an application to protein folding of a mutational variant of the Fyn SH3 domain where (1)H-(15)N dipolar couplings of the invisible unfolded state of the domain are obtained. The approach, which can be used to obtain orientational restraints at other sites in proteins as well, promises to significantly extend the available information necessary for providing a site-specific characterization of structural properties of transient, low populated states that have to this point remained recalcitrant to detailed analysis.
Project description:NMR residual dipolar couplings for the S-peptide of ribonuclease A aligned in C8E5/n-octanol liquid crystals are consistent with the presence of a native-like alpha-helix structure undergoing dynamic fraying. Residues 3-13, which correspond to the first alpha-helix of ribonuclease A, show couplings that become more negative at low temperature and in the presence of salt, conditions which stabilize alpha-helical structure in the S-peptide. By contrast, dipolar couplings from the N and C termini of the peptide are close to zero and remain nearly invariant with changes in solution conditions. Torsion angle dynamics simulations using a gradient of dihedral restraint bounds that increase from the center to the ends of the peptide reproduce the experimentally observed sequence dependence of dipolar couplings. The magnitudes of residual dipolar couplings depend on the anisotropy of the solute. Native proteins often achieve nearly spherical shapes due to the hydrophobic effect. Embryonic partially folded structures such as the S-peptide alpha-helix have an intrinsically greater potential for anisotropy that can result in sizable residual dipolar couplings in the absence of long-range structure.
Project description:Residual dipolar couplings (RDCs) between NC' and NC(alpha) atoms in polypeptide backbones of proteins contain information on the orientation of bond vectors that is complementary to that contained in NH RDCs. The (1)J(NC)(alpha) and (2)J(NC)(alpha) scalar couplings between these atoms also display a Karplus relation with the backbone torsion angles and report on secondary structure. However, these N-C couplings tend to be small and they are frequently unresolvable in frequency domain spectra having the broad lines characteristic of large proteins. Here a TROSY-based J-modulated approach for the measurement of small (15)N-(13)C couplings in large proteins is described. The cross-correlation interference effects inherent in TROSY methods improve resolution and signal to noise ratios for large proteins, and the use of J-modulation to encode couplings eliminates the need to remove frequency distortions from overlapping peaks during data analysis. The utility of the method is demonstrated by measurement of (1)J(NC'), (1)J(NC)(alpha) , and (2)J(NC)(alpha) scalar couplings and (1)D(NC') and D(NC)(alpha) residual dipolar couplings for the myristoylated yeast ARF1.GTPgammas protein bound to small lipid bicelles, a system with an effective molecule weight of approximately 70kDa.
Project description:Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers). However, a rigorous understanding of the relation between a protein's structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules and populates an excited state transiently (about 1?ms) to about 3% at 25?°C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the 'invisible', excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein's function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.
Project description:Nucleic acids undergo structural transitions to access sparsely populated and transiently lived conformational states--or excited conformational states--that play important roles in diverse biological processes. Despite ever-increasing detection of these functionally essential states, 3D structure determination of excited states (ESs) of RNA remains elusive. This is largely due to challenges in obtaining high-resolution structural constraints in these ESs by conventional structural biology approaches. Here, we present nucleic-acid-optimized chemical exchange saturation transfer (CEST) NMR spectroscopy for measuring residual dipolar couplings (RDCs), which provide unique long-range angular constraints in ESs of nucleic acids. We demonstrate these approaches on a fluoride riboswitch, where one-bond (13)C-(1)H RDCs from both base and sugar moieties provide direct structural probes into an ES of the ligand-free riboswitch.
Project description:Phospholamban is an integral membrane protein that controls the calcium balance in cardiac muscle cells. As the function and regulation of this protein require the active involvement of low populated states in equilibrium with the native state, it is of great interest to acquire structural information about them. In this work, we calculate the conformations and populations of the ground state and the three main excited states of phospholamban by incorporating nuclear magnetic resonance residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations. We then provide a description of the manner in which phosphorylation at Ser16 modulates the activity of the protein by increasing the sizes of the populations of its excited states. These results demonstrate that the approach that we describe provides a detailed characterization of the different states of phospholamban that determine the function and regulation of this membrane protein. We anticipate that the knowledge of conformational ensembles enable the design of new dominant negative mutants of phospholamban by modulating the relative populations of its conformational substates.
Project description:An unfolded state ensemble is generated by using a self-avoiding statistical coil model that is based on backbone conformational frequencies in a coil library, a subset of the Protein Data Bank. The model reproduces two apparently contradicting behaviors observed in the chemically denatured state for a variety of proteins, random coil scaling of the radius of gyration and the presence of significant amounts of local backbone structure (NMR residual dipolar couplings). The most stretched members of our unfolded ensemble dominate the residual dipolar coupling signal, whereas the uniformity of the sign of the couplings follows from the preponderance of polyproline II and beta conformers in the coil library. Agreement with the NMR data substantially improves when the backbone conformational preferences include correlations arising from the chemical and conformational identity of neighboring residues. Although the unfolded ensembles match the experimental observables, they do not display evidence of native-like topology. By providing an accurate representation of the unfolded state, our statistical coil model can be used to improve thermodynamic and kinetic modeling of protein folding.
Project description:Bax is known for its pro-apoptotic role within the mitochondrial pathway of apoptosis. However, the mechanism for transitioning Bax from cytosolic to membrane-bound oligomer remains elusive. Previous nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) studies defined monomeric Bax as conformationally homogeneous. Yet it has recently been proposed that monomeric Bax exists in equilibrium with a minor state that is distinctly different from its NMR structure. Here, we revisited the structural analysis of Bax using methods uniquely suited for unveiling "invisible" states of proteins, namely, NMR paramagnetic relaxation enhancements and EPR double electron-electron resonance (DEER). Additionally we examined the effect of glycerol, the co-solvent of choice in DEER studies, on the structure of Bax using NMR chemical-shift perturbations and residual dipolar couplings. Based on our combined NMR and EPR results, Bax is a conformationally homogeneous protein prior to its activation.