Line Shape Analysis of 19F NMR-Monitored Chemical Denaturation of a Fold-Switching Protein RfaH Reveals Its Slow Folding Dynamics.

The recent discovery of metamorphic proteins, which can switch between multiple conformations under native conditions, has challenged the well-established one sequence-one structure paradigm of protein folding. This is exemplified in the C-terminal domain of the multidomain transcription factor RfaH, which converts from an α-helical coiled-coil conformation in its autoinhibited state to a ß-barrel conformation upon activation. Here, we use multisite line shape analysis of 19F NMR-monitored equilibrium chemical denaturation measurements of two 19F-labeled variants of full-length RfaH, to show that it folds/unfolds slowly on the NMR time scale, in an apparent all-or-none fashion at physiological pH and room temperature in the 3.3-4.8 M urea concentration range. The significant thermodynamic stability and slow unfolding rate (kinetic stability) are likely responsible for maintaining the closed autoinhibited state of RfaH, preventing spurious interactions with RNA polymerase (RNAP) when not functional. Our results provide a quantitative understanding of the folding-function relationship in the model fold-switching protein RfaH.

Correlation Between Ambulatory Blood Pressure Monitoring and Office Blood Pressure Measurement in Patients with Hypertension: A Community Study.

BACKGROUND: The current gold standard for blood pressure (BP) measurements is based on office BP measurements (OBPMs) by a sphygmomanometer or a digital device. Ambulatory BP measurement (ABPM) is a noninvasive method for continuous monitoring of BP over a period during routine activities of the patient. Thus, ABPM offers multiple BP readings during the patients' daily routine as compared to the single reading by OBPM at rest. A good correlation exists between mean 24-hour BP readings and the prediction of cardiovascular events. The present multicenter observational study was aimed to assess the correlation between ABPM and OBPM in patients with newly diagnosed, controlled, or uncontrolled hypertension in the community setting. Our hypothesis was to test if ABPM provides any further value in those hypertensive patients in whom the office blood pressure levels are controlled. A supplementary hypothesis was whether obtaining ABPM in patients with newly diagnosed or uncontrolled hypertension yields any value over and beyond OBPM. Another objective was to find out the applicability of ABPM in the community setting where the medical care is provided by primary care family physicians and not by specialists. MATERIALS AND METHODS: Materials and Methods A total of 1000 patients were analysed for this study. Those with controlled hypertension were assigned to Group A, and those with newly diagnosed/untreated hypertension comprised Group B. Group A was followed up during Visit 2 and Group B was followed up during Visit 2 and either Visit 3 or Visit 4 to assess the BP measurements by ABPM and OBPM. RESULTS: The correlation between ABPM and OBPM showed minimal variation in the BP readings of Group A subjects at Visit 2. A variation in BP readings was observed in Group B at Visit 2. Furthermore, the correlation was established between ABPM and OBPM noted for Group B subjects during Visit 3, and minimal variation was noted during Visit 4. CONCLUSIONS: Conclusions A good correlation was observed between ABPM and OBPM during both visits in patients in Groups A and B. However, a notable variation was noted in the diastolic BP readings. Thus, large-scale clinical studies are required to detect the prevalence of hypertension, masked hypertension, and dipping patterns associated with hypertension and other related medical co-morbidities.

Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein.

During pathological aggregation, proteins undergo remarkable conformational re-arrangements to anomalously assemble into a heterogeneous collection of misfolded multimers, ranging from soluble oligomers to insoluble amyloid fibrils. Inspired by fluorescence resonance energy transfer (FRET) measurements of protein folding, an experimental strategy to study site-specific misfolding kinetics during aggregation, by effectively suppressing contributions from inter-molecular FRET, is described. Specifically, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, after the monomeric units transformed into large oligomers OL, which subsequently disaggregated reversibly into small oligomers OS at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of OL and elongation into ß-sheets during the formation of OS. The ß1-α1-ß2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both OL and OS.

Structural mechanisms of oligomer and amyloid fibril formation by the prion protein.

Misfolding and aggregation of the prion protein is responsible for multiple neurodegenerative diseases. Works from several laboratories on folding of both the WT and multiple pathogenic mutant variants of the prion protein have identified several structurally dissimilar intermediates, which might be potential precursors to misfolding and aggregation. The misfolded aggregates themselves are morphologically distinct, critically dependent on the solution conditions under which they are prepared, but always ß-sheet rich. Despite the lack of an atomic resolution structure of the infectious pathogenic agent in prion diseases, several low resolution models have identified the ß-sheet rich core of the aggregates formed in vitro, to lie in the α2-α3 subdomain of the prion protein, albeit with local stabilities that vary with the type of aggregate. This feature article describes recent advances in the investigation of in vitro prion protein aggregation using multiple spectroscopic probes, with particular focus on (1) identifying aggregation-prone conformations of the monomeric protein, (2) conditions which trigger misfolding and oligomerization, (3) the mechanism of misfolding and aggregation, and (4) the structure of the misfolded intermediates and final aggregates.

Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.

The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The ß-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling.

Salt-Mediated Oligomerization of the Mouse Prion Protein Monitored by Real-Time NMR.

The prion protein forms ß-rich soluble oligomers in vitro at pH4 in the presence of physiological concentrations of salt. In the absence of salt, oligomerization and misfolding do not take place in an experimentally tractable timescale. While it is well established that a lowering of pH facilitates misfolding and oligomerization of this protein, the role of salt remains poorly understood. Here, solution-state NMR was used to probe perturbations in the monomeric mouse prion protein structure immediately upon salt addition, prior to the commencement of the oligomerization reaction. The weak binding of salt at multiple sites dispersed all over the monomeric protein causes a weak and non-specific perturbation of structure throughout the protein. The only significant perturbation occurs in the loop between helix 2 and 3 in and around the partially buried K193-E195 salt bridge. The disruption of this key electrostatic interaction is the earliest detectable change in the monomer before any major conformational change occurs and appears to constitute the trigger for the commencement of misfolding and oligomerization. Subsequently, the kinetics of monomer loss, due to oligomerization, was monitored at the individual residue level. The oligomerization reaction was found to be rate-limited by association and not conformational change, with an average reaction order of 2.6 across residues. Not surprisingly, salt accelerated the oligomerization kinetics, in a non-specific manner, by electrostatic screening of the highly charged monomers at acidic pH. Together, these results allowed a demarcation of the specific and non-specific effects of salt on prion protein misfolding and oligomerization.

Cysteine-specific Cu2+ chelating tags used as paramagnetic probes in double electron electron resonance.

Double electron electron resonance (DEER) is an attractive technique that is utilized for gaining insight into protein structure and dynamics via nanometer-scale distance measurements. The most commonly used paramagnetic tag in these measurements is a nitroxide spin label, R1. Here, we present the application of two types of high-affinity Cu(2+) chelating tags, based on the EDTA and cyclen metal-binding motifs as alternative X-band DEER probes, using the B1 immunoglobulin-binding domain of protein G (GB1) as a model system. Both types of tags have been incorporated into a variety of protein secondary structure environments and exhibit high spectral sensitivity. In particular, the cyclen-based tag displays distance distributions with comparable distribution widths and most probable distances within 1-3 Å when compared to homologous R1 distributions. The results display the viability of the cyclen tag as an alternative to the R1 side chain for X-band DEER distance measurements in proteins.

Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag.

Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu(2+) binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC-Cu(2+) K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ~10 Å of the Cu(2+) center experience considerable transverse PREs leading to severely attenuated resonances in 2D (15)N-(13)C correlation spectra. For more distant residues, electron-nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for (15)N-Cu(2+) distances up to ~20 Å.

Protein structure determination with paramagnetic solid-state NMR spectroscopy.

Many structures of the proteins and protein assemblies that play central roles in fundamental biological processes and disease pathogenesis are not readily accessible via the conventional techniques of single-crystal X-ray diffraction and solution-state nuclear magnetic resonance (NMR). On the other hand, many of these challenging biological systems are suitable targets for atomic-level structural and dynamic analysis by magic-angle spinning (MAS) solid-state NMR spectroscopy, a technique that has far less stringent limitations on the molecular size and crystalline state. Over the past decade, major advances in instrumentation and methodology have prompted rapid growth in the field of biological solid-state NMR. However, despite this progress, one challenge for the elucidation of three-dimensional (3D) protein structures via conventional MAS NMR methods is the relative lack of long-distance data. Specifically, extracting unambiguous interatomic distance restraints larger than ∼5 Šfrom through-space magnetic dipole-dipole couplings among the protein (1)H, (13)C, and (15)N nuclei has proven to be a considerable challenge for researchers. It is possible to circumvent this problem by extending the structural studies to include several analogs of the protein of interest, intentionally modified to contain covalently attached paramagnetic tags at selected sites. In these paramagnetic proteins, the hyperfine couplings between the nuclei and unpaired electrons can manifest themselves in NMR spectra in the form of relaxation enhancements of the nuclear spins that depend on the electron-nucleus distance. These effects can be significant for nuclei located up to ∼20 Šaway from the paramagnetic center. In this Account, we discuss MAS NMR structural studies of nitroxide and EDTA-Cu(2+) labeled variants of a model 56 amino acid globular protein, B1 immunoglobulin-binding domain of protein G (GB1), in the microcrystalline solid phase. We used a set of six EDTA-Cu(2+)-tagged GB1 mutants to rapidly determine the global protein fold in a de novo fashion. Remarkably, these studies required quantitative measurements of only approximately four or five backbone amide (15)N longitudinal paramagnetic relaxation enhancements per residue, in the complete absence of the usual internuclear distance restraints. Importantly, this paramagnetic solid-state NMR methodology is general and can be directly applied to larger proteins and protein complexes for which a significant fraction of the signals can be assigned in standard 2D and 3D MAS NMR chemical shift correlation spectra.

High-resolution structure of a protein spin-label in a solvent-exposed ß-sheet and comparison with DEER spectroscopy.

X-ray crystallography has been a useful tool in the development of site-directed spin labeling by resolving rotamers of the nitroxide spin-label side chain in a variety of α-helical environments. In this work, the crystal structure of a doubly spin-labeled N8C/K28C mutant of the B1 immunoglobulin-binding domain of protein G (GB1) was solved. The double mutant formed a domain-swapped dimer under crystallization conditions. Two rotameric states of the spin-label were resolved at the solvent-exposed α-helical site, at residue 28; these are in good agreement with rotamers previously reported for helical structures. The second site, at residue 8 on an interior ß-strand, shows the presence of three distinct solvent-exposed side-chain rotamers. One of these rotamers is rarely observed within crystal structures of R1 sites and suggests that the H(α) and S(δ) hydrogen bond that is common to α-helical sites is absent at this interior ß-strand residue. Variable temperature continuous wave (CW) experiments of the ß-strand site showed two distinct components that were correlated to the rotameric states observed in crystallography. Interestingly, the CW data at room temperature could be fit without the use of an order parameter, which is consistent with the lack of the H(α) and S(δ) interaction. Additionally, double electron electron resonance (DEER) spectroscopy was performed on the GB1 double mutant in its monomeric form and yielded a most probable interspin distance of 25 ± 1 Å. In order to evaluate the accuracy of the measured DEER distance, the rotamers observed in the crystal structure of the domain-swapped GB1 dimer were modeled into a high-resolution structure of the wild type monomeric GB1. The distances generated in the resulting GB1 structural models match the most probable DEER distance within ~2 Å. The results are interesting as they indicate by direct experimental measurement that the rotameric states of R1 found in this crystal provide a very close match to the most probable distance measured by DEER.

Protein fold determined by paramagnetic magic-angle spinning solid-state NMR spectroscopy.

Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solid-state NMR spectroscopy. However, the paucity of distance restraints >5 Å, traditionally derived from measurements of magnetic dipole-dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation efforts. Here, we describe a general approach that enables the rapid determination of global protein fold in the solid phase via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogues of the protein of interest containing covalently attached paramagnetic tags, without the use of conventional internuclear distance restraints. The method is demonstrated using six cysteine-EDTA-Cu(2+) mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone (15)N PREs corresponding to distances of ~10-20 Å were obtained. The mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square deviation of 1.8 Å.

Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of 15N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR.

Magic-angle spinning solid-state NMR measurements of (15)N longitudinal paramagnetic relaxation enhancements (PREs) in (13)C,(15)N-labeled proteins modified with Cu(2+)-chelating tags can yield multiple long-range electron-nucleus distance restraints up to ~20 Å (Nadaud et al. in J Am Chem Soc 131:8108-8120, 2009). Using the EDTA-Cu(2+) K28C mutant of B1 immunoglobulin binding domain of protein G (GB1) as a model, we investigate the effects on such measurements of intermolecular electron-nucleus couplings and intrinsic metal binding sites, both of which may potentially complicate the interpretation of PRE data in terms of the intramolecular protein fold. To quantitatively assess the influence of intermolecular (15)N-Cu(2+) interactions we have determined a nearly complete set of longitudinal (15)N PREs for a series of microcrystalline samples containing ~10, 15 and 25 mol percent of the (13)C,(15)N-labeled EDTA-Cu(2+)-tagged protein diluted in a matrix of diamagnetic natural abundance GB1. The residual intermolecular interactions were found to be minor on the whole and account for only a fraction of the relatively small but systematic deviations observed between the experimental (15)N PREs and corresponding values calculated using protein structural models for residues furthest removed from the EDTA-Cu(2+) tag. This suggests that these deviations are also caused in part by other factors not related to the protein structure, such as the presence in the protein of intrinsic secondary sites capable of binding Cu(2+) ions. To probe this issue we performed a Cu(2+) titration study for K28C-EDTA GB1 monitored by 2D (15)N-(1)H solution-state NMR, which revealed that while for Cu(2+):protein molar ratios of ≤ 1.0 Cu(2+) binds primarily to the high-affinity EDTA tag, as anticipated, at even slightly super-stoichiometric ratios the Cu(2+) ions can also associate with side-chains of aspartate and glutamate residues. This in turn is expected to lead to enhanced PREs for residues located in the vicinity of the secondary Cu(2+) binding sites, and indeed many of these residues were ones found to display the elevated longitudinal (15)N PREs in the solid phase.

Rapid acquisition of multidimensional solid-state NMR spectra of proteins facilitated by covalently bound paramagnetic tags.

We describe a condensed data collection approach that facilitates rapid acquisition of multidimensional magic-angle spinning solid-state nuclear magnetic resonance (SSNMR) spectra of proteins by combining rapid sample spinning, optimized low-power radio frequency pulse schemes and covalently attached paramagnetic tags to enhance protein (1)H spin-lattice relaxation. Using EDTA-Cu(2+)-modified K28C and N8C mutants of the B1 immunoglobulin binding domain of protein G as models, we demonstrate that high resolution and sensitivity 2D and 3D SSNMR chemical shift correlation spectra can be recorded in as little as several minutes and several hours, respectively, for samples containing approximately 0.1-0.2 micromol of (13)C,(15)N- or (2)H,(13)C,(15)N-labeled protein. This mode of data acquisition is naturally suited toward the structural SSNMR studies of paramagnetic proteins, for which the typical (1)H longitudinal relaxation time constants are inherently a factor of at least approximately 3-4 lower relative to their diamagnetic counterparts. To illustrate this, we demonstrate the rapid site-specific determination of backbone amide (15)N longitudinal paramagnetic relaxation enhancements using a pseudo-3D SSNMR experiment based on (15)N-(13)C correlation spectroscopy, and we show that such measurements yield valuable long-range (15)N-Cu(2+) distance restraints which report on the three-dimensional protein fold.

Recent trends in workers' compensation.

Workers' compensation provides cash benefits and medical care to employees who are injured on the job and survivor benefits to the dependents of workers whose deaths result from work-related incidents. Workers' compensation programs in the 50 states and the District of Columbia and federal programs together paid $56.0 billion in medical and cash benefits in 2004, an increase of 2.3 percent over 2003 payments. Of the total, $26.1 billion was for medical care and $29.9 billion was for cash benefits. Employers' costs for workers' compensation in 2004 were $87.4 billion, an increase of 7.0 percent over 2003 spending. Workers' compensation programs and spending vary greatly from state to state. As a source of support for disabled workers, workers' compensation is currently surpassed in size only by Social Security Disability Insurance (DI), which covers impairments of any cause that are significant, long-term impediments to work. Although most recipients of workers' compensation recover and return to work, those with lasting impairments may become eligible for DI benefits, subject to an offset to avoid excessive wage replacement from both programs.

Workers' compensation, Social Security Disability Insurance, and the offset: a fact sheet.

This article offers a brief summary of the workers' compensation and Social Security Disability Insurance programs. Information highlighted includes the differences between the two programs' types and terms of coverage. It compares the differing patterns in workers' compensation and Social Security disability benefits as a percentage of wages over the past few decades and considers the potential causes for such trends. The article also explains the offset provision included in the 1965 Social Security Amendments, the intention behind the offset, and how and when offsets are applied.