Biomedical subjects

David Fushman

Publications and source records attributed to David Fushman.

At least 19 recordsLinked to original sources

PubMed2006

An efficient computational method for predicting rotational diffusion tensors of globular proteins using an ellipsoid representation.

We propose a new computational method for predicting rotational diffusion properties of proteins in solution. The method is based on the idea of representing protein surface as an ellipsoid shell. In contrast to other existing approaches this method uses principal component analysis of protein surface coordinates, which results in a substantial increase in the computational efficiency of the method. Direct comparison with the experimental data as well as with the recent computational approach (Garcia de la Torre; et al. J. Magn. Reson. 2000, B147, 138-146), based on representation of protein surface as a set of small spherical friction elements, shows that the method proposed here reproduces experimental data with at least the same level of accuracy and precision as the other approach, while being approximately 500 times faster. Using the new method we investigated the effect of hydration layer and protein surface topography on the rotational diffusion properties of a protein. We found that a hydration layer constructed of approximately one monolayer of water molecules smoothens the protein surface and effectively doubles the overall tumbling time. We also calculated the rotational diffusion tensors for a set of 841 protein structures representing the known protein folds. Our analysis suggests that an anisotropic rotational diffusion model is generally required for NMR relaxation data analysis in single-domain proteins, and that the axially symmetric model could be sufficient for these purposes in approximately half of the proteins.

Anisotropy↗

PubMed2006

Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection.

15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-(1)H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D(2)O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H(2)O. This analysis revealed a 17-24 degree angle between the NH-bond and the unique axis of the 15N chemical shielding tensor.

Amides↗

PubMed2006

Variability of the 15N chemical shielding tensors in the B3 domain of protein G from 15N relaxation measurements at several fields. Implications for backbone order parameters.

We applied a combination of 15N relaxation and CSA/dipolar cross-correlation measurements at five magnetic fields (9.4, 11.7, 14.1, 16.4, and 18.8 T) to determine the 15N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various model-independent approaches and those based on Lipari-Szabo approximation, all of them yielding similar results. The results indicate a range of site-specific values of the anisotropy (CSA) and orientation of the 15N chemical shielding tensor, similar to those in ubiquitin (Fushman, et al. J. Am. Chem. Soc. 1998, 120, 10947; J. Am. Chem. Soc. 1999, 121, 8577). Assuming a Gaussian distribution of the 15N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02 A NH bond length) and the site-to-site CSA variability is +/-17.6 to +/-21.4 ppm, depending on the method used. This CSA variability is significantly larger than derived previously for ribonuclease H (Kroenke, et al. J. Am. Chem. Soc. 1999, 121, 10119) or recently, using "meta-analysis" for ubiquitin (Damberg, et al. J. Am. Chem. Soc. 2005, 127, 1995). Standard interpretation of 15N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform 15N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the site-specific CSAs obtained from our study removes this discrepancy and allows simultaneous fit of relaxation data at all five fields to Lipari-Szabo spectral densities. These findings emphasize the necessity of taking into account the variability of 15N CSA for accurate analysis of protein dynamics from 15N relaxation measurements.

Amines↗

PubMed2006

Atom-by-atom analysis of global downhill protein folding.

Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.

Entropy↗

PubMed2006

Interdomain mobility in di-ubiquitin revealed by NMR.

Domain orientation and dynamics can play an essential role in the function of multidomain proteins. Lys48-linked polyubiquitin chains, the principal signal for proteasomal protein degradation, adopt a closed conformation at physiological conditions, in which the functionally important residues Leu8, Ile44, and Val70 are sequestered at the interdomain interface. This interface must open in order for these groups to become available for interactions with various chain-recognition factors. Knowledge of the mechanism of domain motion leading to the opening of the interdomain interface in polyubiqutin is, therefore, essential for the understanding of the processes controlling molecular recognition events in polyubiquitin signaling. Here we use NMR to characterize the interdomain dynamics that open the interface in a di-ubiquitin chain. This process occurs via domain reorientations on a 10-ns time scale and with the amplitudes that are sufficient for making functionally important hydrophobic residues in polyubiquitin available for direct interactions with various ubiquitin-binding factors. The analysis revealed the structures of the interconverting conformational states of di-ubiquitin and the rates and amplitudes of this process at near-physiological and acidic pH. The proposed mechanism of domain reorientation is quite general and could serve as a paradigm of interdomain mobility in other multidomain systems.

Hydrogen-Ion Concentration↗

PubMed2006

Analysis of interdomain dynamics in a two-domain protein using residual dipolar couplings together with 15N relaxation data.

In this paper, we propose the idea that simultaneous analysis of NMR relaxation data and residual dipolar couplings (RDCs) can provide information about interdomain dynamics in a multidomain protein, which cannot be derived from each data set separately. Specifically, such an approach can be useful when the interdomain motions occur on a timescale comparable to or slower than the overall tumbling in solution. We analyze residual dipolar couplings together with 15N relaxation data for Lys48-linked di-ubiquitin (Ub2), in which interdomain dynamics are described as interconversion between two distinct conformational states of the protein. Our results show that 15N relaxation and residual dipolar coupling data can be used as two complementary experimental data sets for consistent characterization of interdomain conformations and dynamics in this dual-domain protein.

Models, Molecular↗

PubMed2005

Various strategies of using residual dipolar couplings in NMR-driven protein docking: application to Lys48-linked di-ubiquitin and validation against 15N-relaxation data.

When classical, Nuclear Overhauser Effect (NOE)-based approaches fail, it is possible, given high-resolution structures of the free molecules, to model the structure of a complex in solution based solely on chemical shift perturbation (CSP) data in combination with orientational restraints from residual dipolar couplings (RDCs) when available. RDCs can be incorporated into the docking following various strategies: as direct restraints and/or as intermolecular intervector projection angle restraints (Meiler et al., J Biomol NMR 2000;16:245-252). The advantage of the latter for docking is that they directly define the relative orientation of the molecules. A combined protocol in which RDCs are first introduced as intervector projection angle restraints and at a later stage as direct restraints is shown here to give the best performance. This approach, implemented in our information-driven docking approach HADDOCK (Dominguez et al., J Am Chem Soc 2003;125:1731-1737), is used to determine the solution structure of the Lys48-linked di-ubiquitin, for which chemical shift mapping, RDCs, and (15)N-relaxation data have been previously obtained (Varadan et al., J Mol Biol 2002;324:637-647). The resulting structures, derived from CSP and RDC data, are cross-validated using (15)N-relaxation data. The solution structure differs from the crystal structure by a 20 degrees rotation of the two ubiquitin units relative to each other.

Algorithms↗

PubMed2005

Diverse polyubiquitin interaction properties of ubiquitin-associated domains.

The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class ( approximately 30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.

Cell Extracts↗

PubMed2005

Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain.

Although functional diversity in polyubiquitin chain signaling has been ascribed to the ability of differently linked chains to bind in a distinctive manner to effector proteins, structural models of such interactions have been lacking. Here, we use NMR to unveil the structural basis of selective recognition of Lys48-linked di- and tetraubiquitin chains by the UBA2 domain of hHR23A. Although the interaction of UBA2 with Lys48-linked diubiquitin involves the same hydrophobic surface on each ubiquitin unit as that utilized in monoubiquitin:UBA complexes, our results show how the "closed" conformation of Lys48-linked diubiquitin is crucial for high-affinity binding. Moreover, recognition of Lys48-linked diubiquitin involves a unique epitope on UBA, which allows the formation of a sandwich-like diubiqutin:UBA complex. Studies of the UBA-tetraubiquitin interaction suggest that this mode of UBA binding to diubiquitin is relevant for longer chains.

Amino Acid Sequence↗

PubMed2005

Spin-state selection for increased confidence in cross-correlation rates measurements.

A new approach is described for measuring chemical shift anisotropy (CSA)/dipolar cross-correlated relaxation (CCR) rates based on the selection of the individual 15N doublet components prior to the relaxation period. The method uses the spin-state-selective element (S3E) of Sørensen and co-authors [Meissner et al. (1997) J. Mag. Reson., 128, 92-97]. The main advantage of the new method compared to other J-resolved experiments is that it does not create problems of additional signal overlap encountered in coupled spectra. At the same time, this approach allows a simpler control of magnetization pathways than the indirect methods. The method is demonstrated for the B3 domain of protein G.

Nuclear Magnetic Resonance, Biomolecular↗

PubMed2005

Using NMR spectroscopy to monitor ubiquitin chain conformation and interactions with ubiquitin-binding domains.

Polyubiquitin (polyUb) chains function as signaling molecules that mediate a diverse set of cellular events. The outcome of polyubiquitination depends on the specific linkage between Ub moieties in the chain, and differently linked chains function as distinct intracellular signals. Although an increasing number of Ub-binding proteins that transmit the regulatory information conferred by (poly)ubiquitination have been identified, the molecular mechanisms of linkage-specific signaling and recognition still remain to be understood. Knowledge of the chain structure is expected to provide insights into the basis of diversity in polyUb signaling. Here we describe several NMR approaches aimed at determining the physiological conformation of polyUb and characterization of the chains' interactions with ubiquitin-binding proteins.

Models, Molecular↗

PubMed2004

Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

Polyubiquitin chains: polymeric protein signals.

The 76-residue protein ubiquitin exists within eukaryotic cells both as a monomer and in the form of isopeptide-linked polymers called polyubiquitin chains. In two well-described cases, structurally distinct polyubiquitin chains represent functionally distinct intracellular signals. Recently, additional polymeric structures have been detected in vivo and in vitro, and several large families of proteins with polyubiquitin chain-binding activity have been discovered. Although the molecular mechanisms governing specificity in chain synthesis and recognition are still incompletely understood, the scope of signaling by polyubiquitin chains is likely to be broader than originally envisioned.

Animals↗

PubMed2004

Efficient and accurate determination of the overall rotational diffusion tensor of a molecule from (15)N relaxation data using computer program ROTDIF.

We present a computer program ROTDIF for efficient determination of a complete rotational diffusion tensor of a molecule from NMR relaxation data. The derivation of the rotational diffusion tensor in the case of a fully anisotropic model is based on a six-dimensional search, which could be very time consuming, particularly if a grid search in the Euler angle space is involved. Here, we use an efficient Levenberg-Marquardt algorithm combined with Monte Carlo generation of initial guesses. The result is a dramatic, up to 50-fold improvement in the computational efficiency over the previous approaches. This method is demonstrated on a computer-generated and real protein systems. We also address the issue of sensitivity of the diffusion tensor determination from (15)N relaxation measurements to experimental errors in the relaxation rates and discuss possible artifacts from applying higher-symmetry tensor model and how to recognize them.

Algorithms↗

PubMed2004

Characterization of the overall rotational diffusion of a protein from 15N relaxation measurements and hydrodynamic calculations.

In this chapter, we discuss experimental and theoretical methods for characterizing the overall rotational diffusion of molecules in solution. The methods are illustrated for the B3 domain of protein G, a small protein with rotational anisotropy of Dpar/Dperp = 1.4. The rotational diffusion tensor of the protein is determined directly from 15N relaxation measurements. The experimental data are treated assuming various possible models for the overall tumbling: isotropic, axially symmetric, and fully anisotropic, and the results of these analyses are compared to determine an adequate diffusion model for the protein. These experimentally derived characteristics of the protein are compared with the results of theoretical calculations of the diffusion tensor using various hydrodynamic models, to find optimal models and parameter sets for theoretical predictions. We also derive model-free characteristics of internal backbone motions in the protein, to show that different models for the overall motion can result in significantly different pictures of motion. This emphasizes the necessity of accurately characterizing the overall tumbling of a molecule to determine its local dynamics.

Binding Sites↗

PubMed2003

Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling.

Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules. The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains. Lys48-linked chains act as a universal signal for proteasomal degradation, whereas Lys63-linked chains act as a specific signal in several non-degradative processes. Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking. Here we use NMR methods to determine the structure of a Lys63-linked di-ubiquitin chain. The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu8, Ile44, and Val70 on the ubiquitin units. This structure contrasts with the closed conformation observed for Lys48-linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W. J., Jeffrey, L. C., Carson, M., Zhijian, C., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D. (2002) J. Mol. Biol. 324, 637-647). Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin. In contrast, Lys48-linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined. This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations.

Amino Acid Sequence↗

PubMed2003

Direct measurement of the 15N CSA/dipolar relaxation interference from coupled HSQC spectra.

Here we propose a method for the measurement of the (15)N CSA/dipolar relaxation interference based on direct comparison of the (15)N doublet components observed in a (1)H-coupled (1)H-(15)N HSQC-type spectrum. This allows the determination of the cross-correlation rates with no need for correction factors associated with other methods. The signal overlap problem of coupled HSQC spectra is addressed here by using the IPAP scheme (Ottiger et al., 1998). The approach is applied to the B3 domain of protein G to show that the method provides accurate measurements of the (15)N CSA/dipolar cross-correlation rates.

Anisotropy↗

PubMed2003

Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G.

Because the overall tumbling provides a major contribution to protein spectral densities measured in solution, the choice of a proper model for this motion is critical for accurate analysis of protein dynamics. Here we study the overall and backbone dynamics of the B3 domain of protein G using (15)N relaxation measurements and show that the picture of local motions is markedly dependent on the model of overall tumbling. The main difference is in the interpretation of the elevated R(2) values in the alpha-helix: the isotropic model results in conformational exchange throughout the entire helix, whereas no exchange is predicted by anisotropic models that place the longitudinal axis of diffusion tensor almost parallel to the helix axis. Due to small size (fast tumbling) of the protein, the T(1) values have low sensitivity to NH bond orientation. The diffusion tensor derived from orientation dependence of R(2)/R(1) is anisotropic (D(par)/D(perp)=1.4), with a small rhombic component. In order to distinguish the correct picture of motion, we apply model-independent methods that are sensitive to conformational exchange and do not require knowledge of protein structure or assumptions about its dynamics. A comparison of the CSA/dipolar cross-correlation rate constants with (15)N relaxation rates and the estimation of R(ex) terms from relaxation data at 9.4 and 14.1 T indicate no conformational exchange in the helix, in support of the anisotropic models. The experimentally derived diffusion tensor is in excellent agreement with theoretical predictions from hydrodynamic calculations; a detailed comparison with various hydrodynamic models revealed optimal parameters for hydrodynamic calculations.

Anisotropy↗