Pressure, motion, and conformational entropy in molecular recognition by proteins.

The thermodynamics of molecular recognition by proteins is a central determinant of complex biochemistry. For over a half-century, detailed cryogenic structures have provided deep insight into the energetic contributions to ligand binding by proteins. More recently, a dynamical proxy based on NMR-relaxation methods has revealed an unexpected richness in the contributions of conformational entropy to the thermodynamics of ligand binding. Here, we report the pressure dependence of fast internal motion within the ribonuclease barnase and its complex with the protein barstar. In what we believe is a first example, we find that protein dynamics are conserved along the pressure-binding thermodynamic cycle. The femtomolar affinity of the barnase-barstar complex exists despite a penalty by -TΔSconf of +11.7 kJ/mol at ambient pressure. At high pressure, however, the overall change in side-chain dynamics is zero, and binding occurs with no conformational entropy penalty, suggesting an important role of conformational dynamics in the adaptation of protein function to extreme environments. Distinctive clustering of the pressure sensitivity is observed in response to both pressure and binding, indicating the presence of conformational heterogeneity involving less efficiently packed alternative conformation(s). The structural segregation of dynamics observed in barnase is striking and shows how changes in both the magnitude and the sign of regional contributions of conformational entropy to the thermodynamics of protein function are possible.

Transcriptional Silencing by TsrA in the Evolution of Pathogenic Vibrio cholerae Biotypes.

Vibrio cholerae is a globally important pathogen responsible for the severe epidemic diarrheal disease called cholera. The current and ongoing seventh pandemic of cholera is caused by El Tor strains, which have completely replaced the sixth-pandemic classical strains of V. cholerae To successfully establish infection and disseminate to new victims, V. cholerae relies on key virulence factors encoded on horizontally acquired genetic elements. The expression of these factors relies on the regulatory architecture that coordinates the timely expression of virulence determinants during host infection. Here, we apply transcriptomics and structural modeling to understand how type VI secretion system regulator A (TsrA) affects gene expression in both the classical and El Tor biotypes of V. cholerae We find that TsrA acts as a negative regulator of V. cholerae virulence genes encoded on horizontally acquired genetic elements. The TsrA regulon comprises genes encoding cholera toxin (CT), the toxin-coregulated pilus (TCP), and the type VI secretion system (T6SS), as well as genes involved in biofilm formation. The majority of the TsrA regulon is carried on horizontally acquired AT-rich genetic islands whose loss or acquisition could be directly ascribed to the differences between the classical and El Tor strains studied. Our modeling predicts that the TsrA protein is a structural homolog of the histone-like nucleoid structuring protein (H-NS) oligomerization domain and is likely capable of forming higher-order superhelical structures, potentially with DNA. These findings describe how TsrA can integrate into the intricate V. cholerae virulence gene expression program, controlling gene expression through transcriptional silencing.IMPORTANCE Pathogenic Vibrio cholerae strains express multiple virulence factors that are encoded by bacteriophage and chromosomal islands. These include cholera toxin and the intestinal colonization pilus called the toxin-coregulated pilus, which are essential for causing severe disease in humans. However, it is presently unclear how the expression of these horizontally acquired accessory virulence genes can be efficiently integrated with preexisting transcriptional programs that are presumably fine-tuned for optimal expression in V. cholerae before its conversion to a human pathogen. Here, we report the role of a transcriptional regulator (TsrA) in silencing horizontally acquired genes encoding important virulence factors. We propose that this factor could be critical to the efficient acquisition of accessory virulence genes by silencing their expression until other signals trigger their transcriptional activation within the host.

Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation.

Recent studies suggest that the fast timescale motion of methyl-bearing side chains may play an important role in mediating protein activity. These motions have been shown to encapsulate the residual conformational entropy of the folded state that can potentially contribute to the energetics of protein function. Here, we provide an overview of how to characterize these motions using nuclear magnetic resonance (NMR) spin relaxation methods. The strengths and limitations of several techniques are highlighted in order to assist with experimental design. Particular emphasis is placed on the practical aspects of sample preparation, data collection, data fitting, and statistical analysis. Additionally, discussion of the recently refined "entropy meter" is presented and its use in converting NMR observables to conformational entropy is illustrated. Taken together, these methods should yield new insights into the complex interplay between structure and dynamics in protein function.

Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology.

Pressure and temperature are the two fundamental variables of thermodynamics. Temperature and chemical perturbation are central experimental tools for the exploration of macromolecular structure and dynamics. Though it has long been recognized that hydrostatic pressure offers a complementary and often unique view of macromolecular structure, stability and dynamics, it has not been employed nearly as much. For solution NMR applications the limited use of high-pressure is undoubtedly traced to difficulties of employing pressure in the context of modern multinuclear and multidimensional NMR. Limitations in pressure tolerant NMR sample cells have been overcome and enable detailed studies of macromolecular energy landscapes, hydration, dynamics and function. Here we review the practical considerations for studies of biological macromolecules at elevated pressure, with a particular emphasis on applications in protein biophysics and structural biology.

Anomalous Properties of Lys Residues Buried in the Hydrophobic Interior of a Protein Revealed with 15N-Detect NMR Spectroscopy.

Ionizable residues buried in hydrophobic environments in proteins are essential for many fundamental biochemical processes. These residues titrate with anomalous pKa values that are challenging to reproduce with structure-based calculations owing to the conformational reorganization coupled to their ionization. Detailed characterization of this conformational reorganization is of interest; unfortunately, the properties of buried Lys residues are difficult to study experimentally. Here we demonstrate the utility of 15N NMR spectroscopy to gain insight into the protonation state, state of hydration and conformational dynamics of the Nζ amino group of buried Lys residues. The experiments were applied to five variants of staphylococcal nuclease, with internal Lys residues that titrate with pKa values ranging from 6.2 to 8.1. Direct detection of buried Lys residues with these NMR spectroscopy methods will enable correlation between thermodynamic and structural data as well as unprecedented examination of how conformational transitions coupled to their ionization affect their pKa values.

Entropy in molecular recognition by proteins.

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein-ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein-ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein-ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or "entropy meter" also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water-protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins.

Prognostic Usefulness of the 6-Minute Walk Test in Patients With Severe Aortic Stenosis.

The 6-minute walk test distance (6MWD) has been shown to predict prognosis in selected cohorts of patients with heart failure and outcomes after surgical or transcatheter aortic valve implantation (AVI) in patients with symptomatic severe aortic stenosis (AS). Our objective was to evaluate the association between the 6MWD and outcome in patients with severe AS while remaining under medical treatment. In a prospective observational cohort study, a total of 149 patients diagnosed with severe AS by Doppler echocardiography underwent a 6-minute walk test. The single end point was a composite of all-cause death or hospitalization for heart failure. Patients receiving an AVI were censored from follow-up at the time of their AVI, so that only the events that occurred while the patients remained under medical treatment were included in the analysis. During follow-up (median 12.9 months), the end point occurred in 65 patients (43.6%). Univariate analysis showed an association between the 6MWD and the end point (p <0.001). After adjustment for symptoms, left ventricular ejection fraction, aortic valve area, Charlson co-morbidity score, and anemia, the 6MWD independently predicted the end point (adjusted hazard ratio 0.63; 95% confidence interval 0.45 to 0.89; p = 0.010). The incidence of the composite end point was 12 per 100 patient-years in patients with a 6MWD >331 m compared to 86 per 100 patient-years in those with a 6MWD ≤331 m (p <0.001). In conclusion, although patients with severe AS remain under medical treatment, the 6MWD is independently associated with all-cause death or hospitalization for heart failure.

Evolutionarily Conserved Pattern of Interactions in a Protein Revealed by Local Thermal Expansion Properties.

The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

Effect of internal cavities on folding rates and routes revealed by real-time pressure-jump NMR spectroscopy.

The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site-specific resolution. SNase variants with cavities in the central core of the protein exhibit a highly heterogeneous transition-state ensemble (TSE) with a smaller solvent-excluded void volume than the TSE of the parent SNase. This heterogeneous TSE experiences Hammond behavior, becoming more native-like (higher molar volume) with increasing denaturant concentration. In contrast, the TSE of the L125A variant, which has a cavity at the secondary core, is only slightly different from that of the parent SNase. Because pressure acts mainly to eliminate solvent-excluded voids, which are heterogeneously distributed throughout structures, it perturbs the protein more selectively than chemical denaturants, thereby facilitating the characterization of intermediates and the consequences of packing on folding mechanisms. Besides demonstrating how internal cavities can affect the routes and rates of folding of a protein, this study illustrates how the combination of P-jump and NMR spectroscopy can yield detailed mechanistic insight into protein folding reactions with exquisite site-specific temporal information.

Structural, energetic, and dynamic responses of the native state ensemble of staphylococcal nuclease to cavity-creating mutations.

The effects of cavity-creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C-terminal domain and the interface between α and ß subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the ß-barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions.

Remodeling of the folding free energy landscape of staphylococcal nuclease by cavity-creating mutations.

The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, Δ+PHS. The stabilizing mutations of Δ+PHS enhanced the population of the major folding intermediate. Cavity creation in two different regions of the Δ+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The L125A substitution in the C-terminal helix of Δ+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the Δ+PHS background protein. The I92A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing some states and favoring others, depending upon the mutational context. These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has very profound effects on the roughness of the folding landscape, whereas the effects are less pronounced for an energetically equivalent substitution in an already frustrated region.

Cavities determine the pressure unfolding of proteins.

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

[Clinical determinants and prognostic value of hemoglobin in hospitalized patients with systolic heart failure]. / Determinantes clínicos y valor pronóstico de la hemoglobina en pacientes hospitalizados con insuficiencia cardiaca sistólica.

INTRODUCTION AND OBJECTIVES: Anemia is a common finding in outpatients with heart failure (HF) and is associated with increased mortality. The aims of this study were to identify determinants of the hemoglobin level in a large group of hospitalized patients with systolic HF and to investigate the medium-term prognostic value of the hemoglobin level. METHODS: The study included 460 consecutive patients (age 68.3 [12.3] years, 74% male) who were hospitalized with a diagnosis of HF and left ventricular systolic dysfunction (i.e., a left ventricular ejection fraction <45%). At hospital discharge, biochemical and hematological parameters were measured and clinical and echocardiographic variables were recorded. Patients were followed up for 16.8[9.7] months. RESULTS: Anemia, as defined by World Health Organization criteria, was present in 189 (41.1%) patients. The following independent determinants of the hemoglobin level were identified: age (relative risk [RR]=1.035, 95% CI, 1.011-1.060; P=.004), female sex (RR=1.843, 95% CI, 1.083-3.135; P=.024), diabetes mellitus (RR=1.413, 95% CI, 1.087-1.838; P=.010), plasma urea level (RR=1.013, 95% CI, 1.005-1.022; P=.001), and loop diuretic use (RR=2.801, 95% CI, 1.463-5.364; P=.002). A decrease in hemoglobin level was associated with increased risks of death (RR per g/dL=1.232, 95% CI, 1.103-1.375; P<.001) and death or HF readmission (RR per g/dL=1.152, 95% CI, 1.058-1.255; P<.001), but not with readmission for non-fatal HF (RR per g/dL=1.081, 95% CI, 0.962-1.215; P=.265). Blood transfusion during hospitalization did not alter the increased risk of death (RR=2.19, 95% CI 1.40-3.41; P=.001). CONCLUSIONS: In hospitalized patients with systolic HF, the hemoglobin level at hospital discharge was an independent predictor of death in the medium term, but not of readmission for non-fatal HF. The main determinants of the hemoglobin level were age, sex, renal function, diabetes, and the need for diuretics.

Determinantes clínicos y valor pronóstico de la hemoglobina en pacientes hospitalizados con insuficiencia cardiaca sistólica / Clinical determinants and prognostic value of hemoglobin in hospitalized patients with systolic heart failure

Introducción y objetivos. En pacientes ambulatorios con insuficiencia cardiaca, la anemia es frecuente y se asocia con un aumento de la mortalidad. Estudiamos los determinantes del valor de hemoglobina y su valor pronóstico a medio plazo en una población amplia de pacientes hospitalizados con IC sistólica. Métodos. Se incluyó a 460 pacientes consecutivos (68,3 ± 12,3 años, 74% varones) hospitalizados con el diagnóstico de insuficiencia cardiaca y disfunción sistólica (fracción de eyección del ventrículo izquierdo [FEVI] < 45%). En el momento del alta hospitalaria se realizaron las determinaciones bioquímicas y hematológicas y se recogieron las variables clínicas y ecocardiográficas. Los pacientes fueron seguidos durante 16,8 ± 9,7 meses. Resultados. Un total de 189 (41,1%) pacientes presentaban anemia (según la definición de la Organización Mundial de la Salud). Los determinantes independientes del valor de hemoglobina fueron la edad (riesgo relativo [RR] = 1,035; intervalo de confianza [IC] del 95%, 1,011-1,060; p = 0,004), el sexo femenino (RR = 1,843; IC del 95%, 1,083-3,135; p = 0,024), diabetes mellitus (RR = 1,413; IC del 95%, 1,087-1,838; p = 0,010), urea plasmática (RR = 1,013; IC del 95%, 1,005-1,022; p = 0,001) y diuréticos del asa (RR = 2,801; IC del 995%, 1,463-5,364; p = 0,002). Un menor valor de hemoglobina se asoció con un mayor riesgo de muerte evento (RR = 1,232; IC del 95%, 1,103-1,375; p < 0,001) y del evento combinado de muerte o reingreso por insuficiencia cardiaca (RR = 1,152; IC del 95%, 1,058-1,255; p < 0,001), pero no de reingreso por insuficiencia cardiaca no fatal (RR = 1,081; IC del 95%, 0,962-1,215; p = 0,265). La transfusión de hematíes durante el ingreso no modificó el incremento del riesgo de muerte (RR = 2,19; IC del 95%, 1,40-3,41, p = 0,001). Conclusiones. En pacientes hospitalizados con IC sistólica, el valor de hemoglobina en el momento del alta es un predictor independiente de mortalidad a medio plazo, pero no de reingresos por IC no fatal. Sus principales determinantes fueron la edad, el sexo, la función renal, la diabetes y la necesidad de diuréticos (AU)

Introduction and objectives. Anemia is a common finding in outpatients with heart failure (HF) and is associated with increased mortality. The aims of this study were to identify determinants of the hemoglobin level in a large group of hospitalized patients with systolic HF and to investigate the medium-term prognostic value of the hemoglobin level. Methods. The study included 460 consecutive patients (age 68.3 [12.3] years, 74% male) who were hospitalized with a diagnosis of HF and left ventricular systolic dysfunction (i.e., a left ventricular ejection fraction < 45%). At hospital discharge, biochemical and hematological parameters were measured and clinical and echocardiographic variables were recorded. Patients were followed up for 16.8[9.7] months. Results. Anemia, as defined by World Health Organization criteria, was present in 189 (41.1%) patients. The following independent determinants of the hemoglobin level were identified: age (relative risk [RR]=1.035, 95% CI, 1.011–1.060; P=.004), female sex (RR=1.843, 95% CI, 1.083–3.135; P=.024), diabetes mellitus (RR=1.413, 95% CI, 1.087–1.838; P=.010), plasma urea level (RR=1.013, 95% CI, 1.005–1.022; P=.001), and loop diuretic use (RR=2.801, 95% CI, 1.463–5.364; P=.002). A decrease in hemoglobin level was associated with increased risks of death (RR per g/dL=1.232, 95% CI, 1.103–1.375; P < 001) and death or HF readmission (RR per g/dL=1.152, 95% CI, 1.058–1.255; P < .001), but not with readmission for nonfatal HF (RR per g/dL=1.081, 95% CI, 0.962–1.215; P=.265). Blood transfusion during hospitalization did not alter the increased risk of death (RR=2.19, 95% CI 1.40–3.41; P=.001). Conclusions. In hospitalized patients with systolic HF, the hemoglobin level at hospital discharge was an independent predictor of death in the medium term, but not of readmission for non-fatal HF. The main determinants of the hemoglobin level were age, sex, renal function, diabetes, and the need for diuretics (AU)