Native State Conditions Research Articles

Determining Native-State Dynamics of Mitoneet using Hydrogen Exchange Mass Spectrometry

The protein mitoNEET (mNT) is a protein first described in 2004 that binds the type- 2 diabetes drug pioglitazone. The homodimeric protein is encoded by the gene CISD1 and has been implicated to play still undefined role(s) in type-2 diabetes and Parkinson's Disease. Like all NEET proteins, the mNT monomer possesses a CDGSH domain with three cysteine and one histidine residue coordinating a [2Fe-2S] cluster. Removal of this cluster by treatment with acidic conditions induces mNT unfolding. While there are numerous crystal structures in the Protein Data Bank, little is known about holo mNT dynamics and stability or the unfolding of apo mNT. Here we apply hydrogen exchange mass spectrometry (HXMS) to holo and apo mNT to detect dynamics ranging from local breathing to global unfolding under native-state conditions. With mild shifts in pH and temperature, our N-HXMS approach can detect partially unfolded intermediates, extract the free energy of unfolding and/or the rate constants of unfolding and folding for the native state and intermediates. In addition, we can simultaneously monitor holo and apo states of mNT due to their differences in mass. By systematically comparing their HXMS behavior, we will gain insight into the connection between mNT dynamics and function.

NMR backbone resonance assignments of the prodomain variants of BDNF in the urea denatured state.

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and memory. A single Val66Met variant has been identified in the prodomain of human BDNF that is associated with anxiety, depression and memory disorders. The structural differences within the full-length prodomain Val66 and Met66 isoforms could shed light on the mechanism of action of the Met66 and its impact on the development of neuropsychiatric-associated disorders. In the present study, we report the backbone 1H, 13C, and 15N NMR assignments of both full-length Val66 and Met66 prodomains in the presence of 2M urea. These conditions were utilized to suppress residual structure and aid subsequent native state structural investigations aimed at mapping and identifying variant-dependent conformational differences under native-state conditions.

Abstract 2856: Functional profile of BEZ-235 activity in human tumor primary culture microspheroids by ex vivo analysis of programmed cell death (EVA/PCD)

Abstract Aberrant signaling through the phospho-inositol pathway via the gain of PI3K function or loss of PTEN is a hallmark of many malignancies. Drugs that inhibit this pathway offer novel therapeutic opportunities. The imidazoquinolone derivative, BEZ-235, reversibly inhibits signaling through PI3K & mTOR by competition at ATP binding sites. We used ex vivo analysis of programmed cell death (EVA/PCD) to examine the activity and clinical potential of BEZ 235 in human tumors isolated from 88 surgical tumor specimens. By interrogating drug effects in primary culture micro-spheroids, replete with vascular, stromal and inflammatory elements, the EVA/PCD platform can provide insights into cellular responses under native-state conditions. Lethal concentration 50% values (LC50's), interpolated from dose response curves, were used to compare the activity of BEZ-235 by diagnosis using modified Z-scores. Comparisons between BEZ activity and related inhibitors of PI3K (LY294002), mTOR (Everolimus) & AKT (Phen B15-kindly provided by Dr. Peter Houghton) were conducted by Pearson-Moment. By rank order, BEZ-235 activity revealed upper gastrointestinal, breast, NSCLC and hematological malignancies to have the most favorable profiles, while renal, ovarian, colon and sarcoma specimens fell in the more resistant range. Pearson moments revealed high correlation coefficients (R values) for LY294002=0.56 (P =0.001); Everolimus=0.51 (P= 0.005) & Phen B15=0.89(P= 0.005) all consistent with BEZ-235's known modes of action. As PI3K signaling is associated with inhibition of apoptosis, its up-regulation may confer collateral resistance to other stressors like growth factor withdrawal or cytotoxic drugs. Relationships between BEZ-235 and other classes of drugs are being examined to explore additional correlations and potential combination that could provide future therapeutic opportunities, as will be reported. Supported in part by the Vanguard Cancer Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2856. doi:1538-7445.AM2012-2856

The Short-Time Dynamics of Proteins Near Native State Conditions Signal to Robust Mechanisms Accessible at Long Times

A number of recent computational studies have indicated that a robust pattern of motions is encoded by the particular architecture of a protein. These patterns have been shown to be of relevance to functional motions that occur on the much slower time scale of microseconds or longer. In addition, experimental data derived from NMR relaxation and other motion-containing NMR parameters as well as computational data from MD simulations indicate that the relative movements of residues at short times exhibit discernable correlations with those observed at long times. This intriguing issue was explored in the present work by conducting systematic molecular dynamics simulations of various durations, varying in the range 1 ns ≤ ttot ≤ 0.4 μs. While the observed amplitudes of motions are generally confined to 1 A in short simulations with a general increase to 2-3 A in the long ones, strikingly, the residue displacement distributions (away from equilibrium positions) exhibit comparable patterns. Careful examination of the relationship between the size of observed motion and the duration of simulations revealed a stretched exponential dependence of the the form y μ x0.26, with x as representing the ratio of simulation lengths and y the ratio of the motional amplitudes, consistent with the observations made by Scheraga and coworkers.11. Senet P, Maisuradze GG, Foulie C, Delarue P, Scheraga HA. How main-chains of proteins explore the free-energy landscape in native states. Proc. Natl. Acad. Sci. U. S. A (2008) 105: 19708-19713.

Global Dynamics of Proteins: Bridging Between Structure and Function

Biomolecular systems possess unique, structure-encoded dynamic properties that underlie their biological functions. Recent studies indicate that these dynamic properties are determined to a large extent by the topology of native contacts. In recent years, elastic network models used in conjunction with normal mode analyses have proven to be useful for elucidating the collective dynamics intrinsically accessible under native state conditions, including in particular the global modes of motions that are robustly defined by the overall architecture. With increasing availability of structural data for well-studied proteins in different forms (liganded, complexed, or free), there is increasing evidence in support of the correspondence between functional changes in structures observed in experiments and the global motions predicted by these coarse-grained analyses. These observed correlations suggest that computational methods may be advantageously employed for assessing functional changes in structure and allosteric mechanisms intrinsically favored by the native fold.

Normal mode analysis of biomolecular structures: functional mechanisms of membrane proteins.

Normal mode analysis of biomolecular structures: functional mechanisms of membrane proteins.

A comparative analysis of the equilibrium dynamics of a designed protein inferred from NMR, X-ray, and computations

A detailed analysis of high-resolution structural data and computationally predicted dynamics was carried out for a designed sugar-binding protein. The mean-square deviations in the positions of residues derived from nuclear magnetic resonance (NMR) models and those inferred from X-ray crystallographic B-factors for two different crystal forms were compared with the predictions based on the Gaussian Network Model (GNM) and the results from molecular dynamics (MD) simulations. GNM systematically yielded a higher correlation than MD, with experimental data, suggesting that the lack of atomistic details in the coarse-grained GNM is more than compensated for by the mathematically exact evaluation of fluctuations using the native contacts topology. Evidence is provided that particular loop motions are curtailed by intermolecular contacts in the crystal environment causing a discrepancy between theory and experiments. Interestingly, the information conveyed by X-ray crystallography becomes more consistent with NMR models and computational predictions when ensembles of X-ray models are considered. Less precise (broadly distributed) ensembles indeed appear to describe the accessible conformational space under native state conditions better than B-factors. Our results highlight the importance of using multiple conformations obtained by alternative experimental methods, and analyzing results from both coarse-grained models and atomic simulations, for accurate assessment of motions accessible to proteins under native state conditions.

A Detailed Comparison Between The NMR Ensemble, Two X-ray Models And Computational Predictions Of Motions For A Designed Sugar Binding Protein

Coarse-grained elastic network models with single point representation of amino acids are becoming increasingly popular for describing conformational flexibility and equilibrium dynamics of proteins. In particular, the Gaussian network model (GNM) predictions have been fairly successful in interpreting the residue-level root-mean-square variations in residue positions inferred from NMR ensembles of structural models for a given protein and the fluctuations in residue positions indicated by crystallographic B-factors. Here, we carried out a detailed analysis for a designed sugar binding protein whose structure was solved in two crystal forms by X-ray crystallography and by NMR. Comparison with experimental data and results from molecular dynamics simulations confirm that the GNM predicts well the equilibrium dynamics of this protein and correlates better with the NMR derived data than crystallographic B-factors. The results further stipulate the importance of examining multiple structures determined by different methods as well as performing both analytical and numerical studies, toward gaining an accurate understanding of the type and range of conformational motions accessible to a given protein under native state conditions.

Coupling between global dynamics and signal transduction pathways: a mechanism of allostery for chaperonin GroEL

Despite significant efforts toward understanding the molecular basis of allosteric communication, the mechanisms by which local energetic and conformational changes cooperatively diffuse from ligand-binding sites to distal regions across the 3-dimensional structure of allosteric proteins remain to be established. Recent experimental and theoretical evidence supports the view that allosteric communication is facilitated by the intrinsic ability of the biomolecules to undergo collective changes in structure, triggered by ligand binding. Two groups of studies recently proved to provide insights into such intrinsic, structure-induced effects: elastic network models that permit us to visualize the cooperative changes in conformation that are most readily accessible near native state conditions, and information-theoretic approaches that elucidate the most efficient pathways of signal transmission favored by the overall architecture. Using a combination of these two approaches, we highlight, by way of application to the bacterial chaperonin complex GroEL-GroES, how the most cooperative modes of motion play a role in mediating the propagation of allosteric signals. A functional coupling between the global dynamics sampled under equilibrium conditions and the signal transduction pathways inherently favored by network topology appears to control allosteric effects.

O GNM: online computation of structural dynamics using the Gaussian Network Model

An assessment of the equilibrium dynamics of biomolecular systems, and in particular their most cooperative fluctuations accessible under native state conditions, is a first step towards understanding molecular mechanisms relevant to biological function. We present a web-based system, oGNM that enables users to calculate online the shape and dispersion of normal modes of motion for proteins, oligonucleotides and their complexes, or associated biological units, using the Gaussian Network Model (GNM). Computations with the new engine are 5–6 orders of magnitude faster than those using conventional normal mode analyses. Two cases studies illustrate the utility of oGNM. The first shows that the thermal fluctuations predicted for 1250 non-homologous proteins correlate well with X-ray crystallographic data over a broad range [7.3–15 Å] of inter-residue interaction cutoff distances and the correlations improve with increasing observation temperatures. The second study, focused on 64 oligonucleotides and oligonucleotide–protein complexes, shows that good agreement with experiments is achieved by representing each nucleotide by three GNM nodes (as opposed to one-node-per-residue in proteins) along with uniform interaction ranges for all components of the complexes. These results open the way to a rapid assessment of the dynamics of DNA/RNA-containing complexes. The server can be accessed at .

Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state.

Aggregation of partially folded intermediates populated during protein folding processes has been described for many proteins. Likewise, partially unfolded chains, generated by perturbation of numerous proteins by heat or chemical denaturants, have also been shown to aggregate readily. However, the process of protein aggregation from native-state conditions is less well understood. Granulocyte-colony stimulating factor (G-CSF), a member of the four-helix bundle class of cytokines, is a therapeutically relevant protein involved in stimulating the growth and maturation of phagocytotic white blood cells. Under native-like conditions (37 degrees C [pH 7.0]), G-CSF shows a significant propensity to aggregate. Our data suggest that under these conditions, native G-CSF exists in equilibrium with an altered conformation, which is highly aggregation prone. This species is enriched in 1-2 M GdmCl, as determined by tryptophan fluorescence and increased aggregation kinetics. In particular, specific changes in Trp58 fluorescence report a local rearrangement in the large loop region between helices A and B. However, circular dichroism, reactivity toward cyanylation, and ANS binding demonstrate that this conformational change is subtle, having no substantial disruption of secondary and tertiary structure, reactivity of the free sulfhydryl at Cys17 or exposure of buried hydrophobic regions. There is no indication that this altered conformation is important to biological activity, making it an attractive target for rational protein stabilization.