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1.
Proc Natl Acad Sci U S A ; 114(20): 5171-5176, 2017 05 16.
Article in English | MEDLINE | ID: mdl-28461483

ABSTRACT

An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.


Subject(s)
Bacteriophage Pf1/chemistry , Nuclear Magnetic Resonance, Biomolecular , Pseudomonas aeruginosa/virology , Bacteriophage Pf1/metabolism
2.
Protein Sci ; 23(7): 851-6, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24752984

ABSTRACT

Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Escherichia coli Proteins/chemistry , Hydrolases/chemistry , Lipid Bilayers/chemistry , Magnetic Resonance Spectroscopy/methods , Nanostructures/chemistry , Apolipoproteins/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacteriophage Pf1/genetics , Bacteriophage Pf1/metabolism , Escherichia coli Proteins/metabolism , Hydrogen Bonding , Hydrolases/metabolism , Lipid Bilayers/metabolism , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary
3.
Soft Matter ; 10(10): 1439-49, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24651463

ABSTRACT

Anionic polyelectrolyte filaments are common in biological cells. DNA, RNA, the cytoskeletal filaments F-actin, microtubules, and intermediate filaments, and polysaccharides such as hyaluronan that form the pericellular matrix all have large net negative charge densities distributed over their surfaces. Several filamentous viruses with diameters and stiffnesses similar to those of cytoskeletal polymers also have similar negative charge densities. Extracellular protein filaments such collagen, fibrin and elastin, in contrast, have notably smaller charge densities and do not behave as highly charged polyelectrolytes in solution. This review summarizes data that demonstrate generic counterion-mediated effects on four structurally unrelated biopolymers of similar charge density: F-actin, vimentin, Pf1 virus, and DNA, and explores the possible biological and pathophysiological consequences of the polyelectrolyte properties of biological filaments.


Subject(s)
Actins/metabolism , Bacteriophage Pf1/metabolism , DNA/metabolism , Vimentin/metabolism , Actins/chemistry , Bacteriophage Pf1/chemistry , Biopolymers/chemistry , Biopolymers/metabolism , Body Fluids/chemistry , Body Fluids/metabolism , Cytoskeleton/chemistry , Cytoskeleton/metabolism , DNA/chemistry , Electrolytes/chemistry , Electrolytes/metabolism , Hyaluronic Acid/chemistry , Intermediate Filaments/metabolism , Vimentin/chemistry
4.
J Phys Chem B ; 117(10): 2837-40, 2013 Mar 14.
Article in English | MEDLINE | ID: mdl-23409842

ABSTRACT

Water plays a major structural and functional role around proteins. In an attempt to explore this mechanistic structural aspect of proteins, we present site-specific interaction of hydration water with the major coat protein subunit of filamentous virus Pf1 by magic angle spinning (MAS) solid-state NMR. The interaction of surrounding water with 36 MDa Pf1 virion is investigated in uniformly (13)C, (15)N isotopically labeled; polyethylene glycol precipitated fully hydrated samples by solid-state nuclear magnetic resonance spectroscopy. Dipolar edited two-dimensional (2D) (1)H-(15)N heteronuclear correlation (HETCOR) experiments lead to unambiguous assignments of cross-peaks originating exclusively from (1)H resonances of water molecules correlating to the protein amide nitrogen. An enhanced resolved (1)H chemical shift dimension in these experiments also precludes the need of perdeuteration. We report seven residues spanning the 40-residue continuous α-helical conformation assembly of Pf1 interacting with surrounding water. It shows a highly hydrated inner core inside this viral filamentous assembly. The results obtained also suggest the first evidence of a water-mediated interface cluster formed at the site of Arg44 with the single-stranded DNA genome of the filamentous phage supramolecular assembly.


Subject(s)
Bacteriophage Pf1/chemistry , Capsid Proteins/chemistry , Water/chemistry , Bacteriophage Pf1/metabolism , Capsid Proteins/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Water/metabolism
5.
J Biomol NMR ; 54(1): 53-67, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22828737

ABSTRACT

Main-chain (1)H(N)-(15)N residual dipolar couplings (RDCs) ranging from approximately -200 to 200 Hz have been measured for ubiquitin under strong alignment conditions in Pf1 phage. This represents a ten-fold increase in the degree of alignment over the typical weakly aligned samples. The measurements are made possible by extensive proton-dilution of the sample, achieved by deuteration of the protein with partial back-substitution of labile protons from 25 % H(2)O / 75 % D(2)O buffer. The spectral quality is further improved by application of deuterium decoupling. Since standard experiments using fixed-delay INEPT elements cannot accommodate a broad range of couplings, the measurements were conducted using J-resolved and J-modulated versions of the HSQC and TROSY sequences. Due to unusually large variations in dipolar couplings, the trosy (sharp) and anti-trosy (broad) signals are often found to be interchanged in the TROSY spectra. To distinguish between the two, we have relied on their respective (15)N linewidths. This strategy ultimately allowed us to determine the signs of RDCs. The fitting of the measured RDC values to the crystallographic coordinates of ubiquitin yields the quality factor Q = 0.16, which confirms the perturbation-free character of the Pf1 alignment. Our results demonstrate that RDC data can be successfully acquired not only in dilute liquid crystals, but also in more concentrated ones. As a general rule, the increase in liquid crystal concentration improves the stability of alignment media and makes them more tolerant to variations in sample conditions. The technical ability to measure RDCs under moderately strong alignment conditions may open the door for development of alternative alignment media, including new types of media that mimic biologically relevant systems.


Subject(s)
Deuterium/chemistry , Ubiquitin/chemistry , Bacteriophage Pf1/chemistry , Bacteriophage Pf1/metabolism , Carbon Isotopes/chemistry , Nitrogen Isotopes/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation
6.
Anal Chem ; 84(9): 4063-70, 2012 May 01.
Article in English | MEDLINE | ID: mdl-22472063

ABSTRACT

We present a method for the qualitative and quantitative study of transient metabolic flux of phage infection at the molecular level. The method is based on statistical total correlation spectroscopy (STOCSY) and partial least squares discriminant analysis (PLS-DA) applied to nuclear magnetic resonance (NMR) metabonomic data sets. An algorithm for this type of study is developed and demonstrated. The method has been implemented on (1)H NMR data sets of growth media in planktonic cultures of Pseudomonas aeruginosa infected with bacteriophage pf1. Transient metabolic flux of various important metabolites, identified by STOCSY and PLS-DA analysis applied to the NMR data set, are estimated at various stages of growth. The opportunistic and nosocomial pathogen P. aeruginosa is one of the best-studied model organism for bacterial biofilms. Complete information regarding metabolic connectivity of this system is not possible by conventional spectroscopic approach. Our study presents temporal comparative (1)H NMR metabonomic analyses of filamentous phage pf1 infection in planktonic cultures of P. aeruginosa K strain (PAK). We exemplify here the potential of STOCSY and PLS-DA tools to gain mechanistic insight into subtle changes and to determine the transient flux associated with metabolites following metabolic perturbations resulting from phage infection. Our study has given new avenues in correlating existing postgenomic data with current metabonomic results in P. aeruginosa biofilms research.


Subject(s)
Algorithms , Bacteriophage Pf1/physiology , Host-Pathogen Interactions , Metabolomics/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Pseudomonas aeruginosa/virology , Bacteriophage Pf1/metabolism , Metabolome , Pseudomonas aeruginosa/metabolism
7.
J Phys Chem B ; 115(51): 15406-14, 2011 Dec 29.
Article in English | MEDLINE | ID: mdl-22073926

ABSTRACT

A unified theory for the NMR line shapes of aligned membrane proteins arising from uniaxial disorder (mosaic spread) and global rotational diffusion about the director axis is presented. A superoperator formalism allows one to take into account the effects of continuous radiofrequency irradiation and frequency offsets in the presence of dynamics. A general method based on the Stochastic Liouville Equation makes it possible to bridge the static and dynamic limits in a single model. Simulations of solid-state NMR spectra are performed for a uniform α helix by considering orientational disorder and diffusion of the helix as a whole relative to the alignment axis. The motional narrowing of the resonance lines is highly inhomogeneous and can be used as an additional angular restraint in structure calculations. Experimental solid-state NMR spectra of Pf1 coat protein support the conclusions of the theory for two limiting cases. The static disorder dominates the (15)N NMR spectra of Pf1 aligned on a phage, while fast uniaxial diffusion provides a line narrowing mechanism for the Pf1 protein reconstituted in magnetically aligned bicelles.


Subject(s)
Membrane Proteins/chemistry , Bacteriophage Pf1/metabolism , Capsid Proteins/chemistry , Diffusion , Nitrogen Isotopes/chemistry , Nuclear Magnetic Resonance, Biomolecular
8.
Eur Biophys J ; 40(3): 221-34, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21082179

ABSTRACT

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.


Subject(s)
Bacteriophage Pf1/chemistry , Magnetic Resonance Spectroscopy/methods , X-Ray Diffraction/methods , Bacteriophage Pf1/metabolism , Capsid/chemistry , Capsid Proteins/chemistry , Cell Membrane/chemistry , Models, Molecular , Protein Conformation , Pseudomonas/virology , Viral Proteins/chemistry , Virion/chemistry
9.
Biophys J ; 99(5): 1465-74, 2010 Sep 08.
Article in English | MEDLINE | ID: mdl-20816058

ABSTRACT

The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16-A46) around its long axis changes dramatically (by 160 degrees) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2-G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state (1)H/(15)N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.


Subject(s)
Bacteriophage Pf1/physiology , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cell Membrane/metabolism , Virus Assembly , Bacteriophage Pf1/metabolism , Lipid Bilayers/metabolism , Micelles , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Solutions
10.
J Biol Chem ; 285(47): 37051-9, 2010 Nov 19.
Article in English | MEDLINE | ID: mdl-20736177

ABSTRACT

Magic angle spinning solid-state NMR has been used to study the structural changes in the Pf1 filamentous bacteriophage, which occur near 10 °C. Comparisons of NMR spectra recorded above and below 10 °C reveal reversible perturbations in many NMR chemical shifts, most of which are assigned to atoms of hydrophobic side chains of the 46-residue subunit. The changes mainly involve groups located in patches on the interfaces between neighboring capsid subunits. The observations show that the transition adjusts the hydrophobic interfaces between fairly rigid subunits. The low temperature form has been generally more amenable to structure determination; spin diffusion experiments on this form revealed unambiguous contacts between side chains of neighboring subunits. These contacts are important constraints for structure modeling.


Subject(s)
Bacteriophage Pf1/metabolism , Hydrophobic and Hydrophilic Interactions , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy , Pseudomonas aeruginosa/metabolism , Bacteriophage Pf1/chemistry , Pseudomonas aeruginosa/virology
11.
J Magn Reson ; 189(2): 157-65, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17900951

ABSTRACT

In NMR spectra of complex proteins, sparse isotope enrichment can be important, in that the removal of many (13)C-(13)C homonuclear J-couplings can narrow the lines and thereby facilitate the process of spectral assignment and structure elucidation. We present a simple scheme for selective yet extensive isotopic enrichment applicable for production of proteins in organisms utilizing the Entner-Doudoroff (ED) metabolic pathway. An enrichment scheme so derived is demonstrated in the context of a magic-angle spinning solid-state NMR (MAS SSNMR) study of Pf1 bacteriophage, the host of which is Pseudomonas aeruginosa, strain K (PAK), an organism that uses the ED pathway for glucose catabolism. The intact and infectious Pf1 phage in this study was produced by infected PAK cells grown on a minimal medium containing 1-(13)C d-glucose ((13)C in position 1) as the sole carbon source, as well as (15)NH(4)Cl as the only nitrogen source. The 37MDa Pf1 phage consists of about 93% major coat protein, 1% minor coat proteins, and 6% single-stranded, circular DNA. As a consequence of this composition and the enrichment scheme, the resonances in the MAS SSNMR spectra of the Pf1 sample were almost exclusively due to carbonyl carbons in the major coat protein. Moreover, 3D heteronuclear NCOCX correlation experiments also show that the amino acids leucine, serine, glycine, and tyrosine were not isotopically enriched in their carbonyl positions (although most other amino acids were), which is as expected based upon considerations of the ED metabolic pathway. 3D NCOCX NMR data and 2D (15)N-(15)N data provided strong verification of many previous assignments of (15)N amide and (13)C carbonyl shifts in this highly congested spectrum; both the semi-selective enrichment patterns and the narrowed linewidths allowed for greater certainty in the assignments as compared with use of uniformly enriched samples alone.


Subject(s)
Algorithms , Bacteriophage Pf1/metabolism , Gene Expression Profiling/methods , Magnetic Resonance Spectroscopy/methods , Pseudomonas aeruginosa/virology , Viral Proteins/metabolism , Carbon Isotopes
12.
J Biol Chem ; 281(39): 28831-6, 2006 Sep 29.
Article in English | MEDLINE | ID: mdl-16893883

ABSTRACT

The plant homeodomain (PHD) zinc finger is one of 14 known zinc-binding domains. PHD domains have been found in more than 400 eukaryotic proteins and are characterized by a Cys(4)-His-Cys(3) zinc-binding motif that spans 50-80 residues. The precise function of PHD domains is currently unknown; however, the PHD domains of the ING1 and ING2 tumor suppressors have been shown recently to bind phosphoinositides (PIs). We have recently identified a novel PHD-containing protein, Pf1, as a binding partner for the abundant and ubiquitous transcriptional corepressor mSin3A. Pf1 contains two PHD zinc fingers, PHD1 and PHD2, and functions to bridge mSin3A to the TLE1 corepressor. Here, we show that PHD1, but not PHD2, binds several monophosporylated PIs but most strongly to PI(3)P. Surprisingly, a polybasic region that follows the PHD1 is necessary for PI(3)P binding. Furthermore, this polybasic region binds specifically to PI(3)P when fused to maltose-binding protein, PHD2, or as an isolated peptide, demonstrating that it is sufficient for specific PI binding. By exchanging the polybasic regions between different PHD fingers we show that this region is a strong determinant of PI binding specificity. These findings establish the Pf1 polybasic region as a phosphoinositide-binding module and suggest that the PHD domains function down-stream of phosphoinositide signaling triggered by the interaction between polybasic regions and phosphoinositides.


Subject(s)
Bacteriophage Pf1/metabolism , Amino Acid Motifs , Amino Acid Sequence , Molecular Sequence Data , Peptides/chemistry , Phosphatidylinositols/chemistry , Phosphorylation , Plant Proteins/chemistry , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Signal Transduction , Zinc Fingers
13.
J Am Chem Soc ; 127(43): 15032-3, 2005 Nov 02.
Article in English | MEDLINE | ID: mdl-16248635

ABSTRACT

The measurement of independent sets of NMR residual dipolar couplings (RDCs) in multiple alignment media can provide a detailed view of biomolecular structure and dynamics, yet remains experimentally challenging. It is demonstrated here that independent sets of RDCs can be measured for ubiquitin using just a single alignment medium composed of aligned bacteriophage Pf1 particles embedded in a strained polyacrylamide gel matrix. Using this composite medium, molecular alignment can be modulated by varying the angle between the directors of ordering for the Pf1 and strained gel matrix, or by varying the ionic strength or concentration of the Pf1 particles. This approach offers significant advantages in that greater experimental control can be exercised over the acquisition of multi-alignment RDC data while a homogeneous chemical environment is maintained across all of the measured RDC data.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Proteins/chemistry , Bacteriophage Pf1/metabolism , Electrophoresis, Polyacrylamide Gel , Protein Conformation , Proteins/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism
14.
J Mol Biol ; 341(3): 869-79, 2004 Aug 13.
Article in English | MEDLINE | ID: mdl-15288792

ABSTRACT

The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.


Subject(s)
Bacteriophage Pf1/metabolism , Capsid Proteins/chemistry , Magnetic Resonance Spectroscopy/methods , Bacteriophages/metabolism , Neutrons , Protein Conformation , Protein Structure, Tertiary , Software , Statistics as Topic , Temperature , X-Ray Diffraction , X-Rays
16.
Biophys Chem ; 107(3): 255-62, 2004 Feb 15.
Article in English | MEDLINE | ID: mdl-14967240

ABSTRACT

Partially resolved 17O NMR quintet was observed in a filamentous bacteriophage Pf1 solution at 70 degrees C with a quadrupole splitting approximately 100 Hz. As the temperature decreased, the resolution was reduced but the line shapes were still indicative of residual quadrupole splitting. Line shape analyses were performed using the quadrupolar relaxation theory for spin 5/2. The contribution to the residual quadrupole splitting from the electric field gradients stemming from the phage filaments, which were oriented in the magnet, was taken into account. As a result, the observed 17O spectra at different temperatures were simulated and the hydration number of the phage DNA was determined.


Subject(s)
Bacteriophage Pf1/chemistry , Water/analysis , Bacteriophage Pf1/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Nuclear Magnetic Resonance, Biomolecular/methods , Oxygen Isotopes , Pseudomonas aeruginosa/chemistry , Solutions , Temperature , Thermodynamics
17.
J Biomol NMR ; 20(4): 365-77, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11563559

ABSTRACT

The phase diagram of Pf1 solutions has been studied indirectly by observation of 2H quadrupole splittings of the solvent signal and measurement of dipolar couplings in solute macromolecules. At low volume fractions of Pf1 and at high ionic strength, alignment of both the phage and the solute depends strongly on the strength of the magnetic field. Both the theoretical and experimentally determined phase diagram of Pf1 show that at low concentrations and high ionic strengths the solution becomes isotropic. However, just below the nematic phase boundary the behavior of the system is paranematic, with cooperative alignment which depends on the strength of the applied magnetic field. Above 16 mg/ml Pf1 is fully nematic up to 600 mM NaCl. Alignment of proteins with a significant electric dipole moment, which tends to be strong in Pf1, can be reduced by either high ionic strength or low phage concentration. Because ionic strength modulates both the orientation and magnitude of the alignment tensor in Pf1 medium, measurement at two ionic strengths can yield linearly independent alignment tensors.


Subject(s)
Bacteriophage Pf1/chemistry , Water/chemistry , Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacteriophage Pf1/metabolism , Carbon Radioisotopes/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Humans , Nitrogen Radioisotopes/chemistry , Nuclear Magnetic Resonance, Biomolecular , Osmolar Concentration , Protons , Solutions , Temperature , Ubiquitin/chemistry , Ubiquitin/metabolism
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