Internal motions in biomolecules studied by NMR spectroscopy:an application to major urinary protein-1 and its complex with 2-methoxy-3-isobutylpyrazine

Since many biological processes occur on the μs to ms time scale, internal dynamics on that time scale is believed to be related to biological functionality. The characterization of internal dynamics is thus an important issue to improve our understanding of the biological activity of macromolecules, such as proteins, RNA and DNA fragments. Solution NMR spectroscopy, in particular relaxation measurements, is well suited for the determination of exchange rates on that slow time scale. The formation of a small pheromone-protein complex is associated with some kind of internal rearrangements, necessary to accomodate the ligand. In this work, we are interested in the changes in internal dynamic upon the binding of a model odorant molecule, the 2-methoxy-3-isobutylpyrazine, to Major Urinary Protein I, MUP I. The study of the internal dynamics of both apo- and holo-MUP I has been carried out with the goal of monitoring the differences between the two states of the protein. In fact, impressive changes in chemical shifts are present between the two forms, indicating that some internal rearrangements have taken place. New and more classical experiments have been applied to MUP I in order to gain insight into the different types of motions that occurs at different time scales. The presence of chemical exchange has been identiτed in apo-MUP I using the classical Carr-Purcell-Meiboom-Gill experiment on single quantum 15N magnetisation. Surprisly, in holo-MUP I no presence of chemical exchange has been found, so in which time scale are the dynamics which leads to the release of the ligand in the environnment? Slow motions (ms-μs) in both apo- and holo-MUP I have been found using a new experiment, which highlight the presence of correlated motions in the backbone of proteins

Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308K, exchange rates k ex=500-2000s−1 were typically observed in APO-rMUP for residues located adjacent to a β-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local ‘breathing' motions rather than a concerted global conformational chang

Evidence of Slow Motions by Cross-Correlated Chemical Shift Modulation in Deuterated and Protonated Proteins

Cross-correlated fluctuations of isotropic chemical shifts can provide evidence for slow motions in biomolecules. Slow side-chain dynamics have been investigated in 15N and 13C enriched ubiquitin by monitoring the relaxation of Cα-Cβ two-spin coherences (Frueh etal., 2001). This method, which had hitherto been demonstrated only for protonated ubiquitin, has now been applied to both protonated and deuterated proteins. Deuteration reduces the dipole-dipole contributions to the DD/DD cross-correlation, thus facilitating the observation of subtle effects due to cross-correlation of the fluctuations of the isotropic 13C chemical shifts. The decays of double- and zero-quantum coherences are significantly slower in the deuterated protein than in the protonated sample. Slow motions are found both in loops and in secondary structure element

Effects of Protein-pheromone Complexation on Correlated Chemical Shift Modulations

Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titration calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavourable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chemical shifts of carbonyl C′ and amide N nuclei. Correlated chemical shift modulations (CSM/CSM) in MUP have been determined by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C′N} and DQC{C′N}, and by accounting for the effects of correlated fluctuations of dipole-dipole couplings (DD/DD) and chemical shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C′ and N nuclei that have been determined in earlier work. The effects of complexation on slow time-scale protein dynamics can be determined by comparing the temperature dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand

In vivo H-1 NMR measurement of glycine in rat brain at 9.4 T at short echo time

Glycine is an amino acid present in mammalian brain, where it acts as an inhibitory and excitatory neurotransmitter. The two detectable protons of glycine give rise to a singlet at 3.55 ppm that overlaps with the more intense myo-inositol resonances, and its measurement has traditionally required specific editing efforts. The aim of the current study was to reduce the signal intensity of myo-inositol relative to that of glycine by exploiting the fast signal J-evolution of the myo-inositol spin system when using a single spin-echo localization method we recently introduced. Glycine was detected at TE = 20 ms with an average Cramer-Rao lower bound (CRLB) of 8.6% +/- 1.5% in rat brain (N = 5), at 9.4 T. The concentration of glycine was determined using LCModel analysis at 1.1 +/- 0.1 mM, in good agreement with biochemical measurements previously reported. We conclude that at high magnetic fields, glycine can be measured at a relatively short echo time (TE) without additional editing efforts

Effects of Protein-pheromone Complexation on Correlated Chemical Shift Modulations

Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titrn. calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavorable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chem. shifts of carbonyl C' and amide N nuclei. Correlated chem. shift modulations (CSM/CSM) in MUP have been detd. by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C'N} and DQC{C'N}, and by accounting for the effects of correlated fluctuations of dipole-dipole couplings (DD/DD) and chem. shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C' and N nuclei that have been detd. in earlier work. The effects of complexation on slow time-scale protein dynamics can be detd. by comparing the temp. dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand). [on SciFinder (R)

Ventricular arrhythmias in young competitive athletes: Prevalence, determinants, and underlying substrate

Whether ventricular arrhythmias (VAs) represent a feature of the adaptive changes of the athlete's heart remains elusive. We aimed to assess the prevalence, determinants, and underlying substrates of VAs in young competitive athletes.Background--Whether ventricular arrhythmias (VAs) represent a feature of the adaptive changes of the athlete's heart remains elusive. We aimed to assess the prevalence, determinants, and underlying substrates of VAs in young competitive athletes. Method and Results--We studied 288 competitive athletes (age range, 16-35 years; median age, 21 years) and 144 sedentary individuals matched for age and sex who underwent 12-lead 24-hour ambulatory electrocardiographic monitoring. VAs were evaluated in terms of number, complexity (ie, couplet, triplet, or nonsustained ventricular tachycardia), exercise inducibility, and morphologic features. Twenty-eight athletes (10%) and 13 sedentary individuals (11%) showed > 10 isolated premature ventricular beats (PVBs) or 651 complex VA (P=0.81). Athletes with > 10 isolated PVBs or 651 complex VA were older (median age, 26 versus 20 years; P=0.008) but did not differ with regard to type of sport, hours of training, and years of activity compared with the remaining athletes. All athletes with > 10 isolated PVBs or 651 complex VA had a normal echocardiographic examination; 17 of them showing > 500 isolated PVBs, exercise-induced PVBs, and/or complex VA underwent additional cardiac magnetic resonance, which demonstrated nonischemic left ventricular late gadolinium enhancement in 3 athletes with right bundle branch block PVBs morphologic features. Conclusions--The prevalence of > 10 isolated PVBs or 651 complex VA at 24-hour ambulatory electrocardiographic monitoring did not differ between young competitive athletes and sedentary individuals and was unrelated to type, intensity, and years of sports practice. An underlying myocardial substrate was uncommon and distinctively associated with right bundle branch block VA morphologic features

Morphofunctional abnormalities of mitral annulus and arrhythmic mitral valve prolapse

Background\u2014Arrhythmic mitral valve prolapse (MVP) is characterized by myxomatous leaflets and left ventricular (LV) fibrosis of papillary muscles and inferobasal wall. We searched for morphofunctional abnormalities of the mitral valve that could explain a regional mechanical myocardial stretch. Methods and Results\u2014Thirty-six (27 female patients; median age: 44 years) arrhythmic MVP patients with LV late gadolinium enhancement on cardiac magnetic resonance and no or trivial mitral regurgitation, and 16 (6 female patients; median age: 40 years) MVP patients without LV late gadolinium enhancement were investigated by morphofunctional cardiac magnetic resonance. Mitral annulus disjunction (median: 4.8 versus 1.8 mm; P1.5 (22 [61%] versus 4 [25%]; P=0.016) were higher in MVP patients with late gadolinium enhancement than in those without. A linear correlation was found between mitral annulus disjunction and curling (R=0.85). A higher prevalence of auscultatory midsystolic click (26 [72%] versus 6 [38%]; P=0.018) was also noted. Histology of the mitral annulus showed a longer mitral annulus disjunction in 50 sudden death patients with MVP and LV fibrosis than in 20 patients without MVP (median: 3 versus 1.5 mm; P<0.001). Conclusions\u2014Mitral annulus disjunction is a constant feature of arrhythmic MVP with LV fibrosis. The excessive mobility of the leaflets caused by posterior systolic curling accounts for a mechanical stretch of the inferobasal wall and papillary muscles, eventually leading to myocardial hypertrophy and scarring. These mitral annulus abnormalities, together with auscultatory midsystolic click, may identify MVP patients who would need arrhythmic risk stratification

Atrial electro-functional predictors of incident atrial fibrillation in cardiac amyloidosis

Background: Atrial fibrillation (AF) is common in patients with cardiac amyloidosis (CA) and is a significant risk factor for heart failure hospitalization and thromboembolic events. Objective: to investigate the atrial electro-functional predictors of incident AF in CA. Methods: A multicenter, observational study performed in 4 CA referral centers including sinus rhythm patients with light-chain (AL) and transthyretin (ATTR) CA undergoing electrocardiogram (ECG) and cardiac magnetic resonance (CMR). The primary endpoint was new-onset AF occurrence. Results: Overall, 96 patients (AL-CA: n=40; ATTR-CA n=56) were enrolled. During an 18-month median follow-up (Q1-Q3:7-29), 30 patients (29%) had incident AF. Compared with those without, patients with AF were older (79 vs 73 years, p=0.001) and more frequently with ATTR (73% vs 27%, p78 years showed a cumulative incidence for AF of 40% at 12 months. This risk was significantly higher than that carried by one (8.5%) or none (7.6%) of these three risk factors. Conclusions: In patients with CA, older age, IAB on 12-lead ECG and reduced LAEF on CMR are significant and independent predictors of incident AF. A closer screening for AF is advisable in CA patients carrying these features