Probing amino acid side chains of the integral membrane protein PagP by solution NMR: Side chain immobilization facilitates association of secondary structures.

Solution NMR spectroscopy of large protein systems is hampered by rapid signal decay, so most multidimensional studies focus on long-lived 1H-13C magnetization in methyl groups and/or backbone amide 1H-15N magnetization in an otherwise perdeuterated environment. Herein we demonstrate that it is possible to biosynthetically incorporate additional 1H-12C groups that possess long-lived magnetization using cost-effective partially deuterated or unlabeled amino acid precursors added to Escherichia coli growth media. This approach is applied to the outer membrane enzyme PagP in membrane-mimetic dodecylphosphocholine micelles. We were able to obtain chemical shift assignments for a majority of side chain 1H positions in PagP using nuclear Overhauser enhancements (NOEs) to connect them to previously assigned backbone 1H-15N groups and newly assigned 1H-13C methyl groups. Side chain methyl-to-aromatic NOEs were particularly important for confirming that the amphipathic α-helix of PagP packs against its eight-stranded ß-barrel, as indicated by previous X-ray crystal structures. Interestingly, aromatic NOEs suggest that some aromatic residues in PagP that are buried in the membrane bilayer are highly mobile in the micellar environment, like Phe138 and Phe159. In contrast, Tyr87 in the middle of the bilayer is quite rigid, held in place by a hydrogen bonded network extending to the surface that resembles a classic catalytic triad: Tyr87-His67-Asp61. This hydrogen bonded arrangement of residues is not known to have any catalytic activity, but we postulate that its role is to immobilize Tyr87 to facilitate packing of the amphipathic α-helix against the ß-barrel.

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction.

Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant KD of 6-24 µM, stabilizing the activated complex between troponin C and the switch region of troponin I. The interaction between RPI-194 and troponin C is weak (KD 311 µM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 of isometric contraction from 6.28 to 6.99 in mouse slow skeletal muscle fibers and from 5.68 to 5.96 in skinned cardiac trabeculae at 100 µM concentration. There is also some cross-reactivity with fast skeletal muscle fibers (pCa50 increases from 6.27 to 6.52). In the slack test performed on the same skinned skeletal muscle fibers, RPI-194 slowed the velocity of unloaded shortening at saturating calcium concentrations, suggesting that it slows the rate of actin-myosin cross-bridge cycling under these conditions. However, RPI-194 had no effect on the ATPase activity of purified actin-myosin. In isolated unloaded mouse cardiomyocytes, RPI-194 markedly decreased the velocity and amplitude of contractions. In contrast, cardiac function was preserved in mouse isolated perfused working hearts. In summary, the novel troponin activator RPI-194 acts as a calcium sensitizer in all striated muscle types. Surprisingly, it also slows the velocity of unloaded contraction, but the cause and significance of this is uncertain at this time. RPI-194 represents a new class of non-specific troponin activator that could potentially be used either to enhance cardiac muscle contractility in the setting of systolic heart failure or to enhance skeletal muscle contraction in neuromuscular disorders.

Dilated Cardiomyopathy Mutations and Phosphorylation disrupt the Active Orientation of Cardiac Troponin C.

Cardiac troponin (cTn) is made up of three subunits, cTnC, cTnI, and cTnT. The regulatory N-terminal domain of cTnC (cNTnC) controls cardiac muscle contraction in a calcium-dependent manner. We show that calcium-saturated cNTnC can adopt two different orientations, with the "active" orientation consistent with the 2020 cryo-EM structure of the activated cardiac thin filament by Yamada et al. Using solution NMR 15N R2 relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R2 10 s-1), being connected by a flexible linker. However, upon addition of cTnI1-77, the complex tumbles as a rigid unit (R2 30 s-1). cTnI phosphomimetic mutants S22D/S23D, S41D/S43D and dilated cardiomyopathy- (DCM-)associated mutations cTnI K35Q, cTnC D75Y, and cTnC G159D destabilize the active orientation of cNTnC, with intermediate 15N R2 rates (R2 17-23 s-1). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI1-37 and cTnI135-209. Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI38-134, cTnC-cTnT223-288, or cTnC-cTnI38-134-cTnT223-288), the cNTnC domain is still immobilized, revealing a new interaction between cNTnC and the IT-arm that stabilizes a "dormant" orientation. We propose that the calcium sensitivity of the cardiac troponin complex is regulated by an equilibrium between active and dormant orientations, which can be shifted through post-translational modifications or DCM-associated mutations.

Structure and proteolytic susceptibility of the inhibitory C-terminal tail of cardiac troponin I.

BACKGROUND: Cardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal tail, cTnI135-209, binds actin to shut off cardiac muscle contraction, whereas the competing calcium-dependent binding of the switch region, cTnI146-158, by cardiac troponin C (cTnC) triggers contraction. The N-terminal tail, cTnI1-37, regulates the calcium affinity of cTnC. cTnI is known to be susceptible to proteolytic cleavage by matrix metalloproteinase-2 (MMP-2) and calpain, two intracellular proteases implicated in ischemia-reperfusion injury. METHODS: Soluble fragments of cTnI containing its N- and C-terminal tails, cTnI1-77 and cTnI135-209, were highly expressed and purified from E. coli. We performed in vitro proteolysis studies of both constructs using liquid chromatography-mass spectrometry and solution NMR studies of the C-terminal tail. RESULTS: cTnI135-209 is intrinsically disordered, though it contains three regions with helical propensity (including the switch region) that acquire more structure upon actin binding. We identified three precise MMP-2 cleavage sites at cTnI P17-I18, A156-L157, and G199-M200. In contrast, calpain-2 has numerous cleavage sites throughout Y25-T30 and A152-A160. The critical cTnI switch region is targeted by both proteases. CONCLUSIONS: Both N-terminal and C-terminal tails of cTnI are susceptible to cleavage by MMP-2 and calpain-2. Binding to cTnC or actin confers some protection to proteolysis, which can be understood in terms of their interactions as probed by NMR studies. GENERAL SIGNIFICANCE: cTnI is an important marker of intracellular proteolysis in cardiomyocytes, given its many protease-specific cut sites, high natural abundance, indispensable functional role, and clinical use as gold standard biomarker of myocardial injury.

The calcium sensitizer drug MCI-154 binds the structural C-terminal domain of cardiac troponin C.

The compound MCI-154 was previously shown to increase the calcium sensitivity of cardiac muscle contraction. Using solution NMR spectroscopy, we demonstrate that MCI-154 interacts with the calcium-sensing subunit of the cardiac troponin complex, cardiac troponin C (cTnC). Surprisingly, however, it binds only to the structural C-terminal domain of cTnC (cCTnC), and not to the regulatory N-terminal domain (cNTnC) that determines the calcium sensitivity of cardiac muscle. Physiologically, cTnC is always bound to cardiac troponin I (cTnI), so we examined its interaction with MCI-154 in the presence of two soluble constructs, cTnI1-77 and cTnI135-209, which contain all of the segments of cTnI known to interact with cTnC. Neither the cTnC-cTnI1-77 complex nor the cTnC-cTnI135-209 complex binds to MCI-154. Since residues 39-60 of cTnI are known to bind tightly to the cCTnC domain to form a structured core that is invariant throughout the cardiac cycle, we conclude that MCI-154 does not bind to cTnC when it is part of the intact cardiac troponin complex. Thus, MCI-154 likely exerts its calcium sensitizing effect by interacting with a target other than cardiac troponin.

Stereoselective Deuteration in Aspartate, Asparagine, Lysine, and Methionine Amino Acid Residues Using Fumarate as a Carbon Source for Escherichia coli in D2O.

Perdeuteration with selective 1H,13C-enrichment of methyl groups has enabled solution NMR studies of large (>30 kDa) protein systems. However, we propose that for all non-methyl positions, only magnetization originating from 1H-12C groups is sufficiently long-lived, and it can be transferred via through-space NOEs to slowly relaxing 1H-15N or 1H-13C methyl groups to achieve multidimensional solution NMR. We demonstrate stereoselective 1H,12C-labeling by adding relatively inexpensive unlabeled carbon sources to Escherichia coli growth media in D2O. Using our model system, a mutant WW domain from human Pin1, we compare deuteration patterns in 19 amino acids (all except cysteine). Protein grown using glucose as the sole carbon source had high levels of protonation in aromatic rings and the Hß positions of serine and tryptophan. In contrast, using our FROMP media (fumarate, rhamnose, oxalate, malonate, pyruvate), stereoselective protonation of Hß2 with deuteration at Hα and Hß3 was achieved in Asp, Asn, Lys, and Met residues. In solution NMR, stereospecific chemical shift assignments for Hß are typically obtained in conjunction with χ1 dihedral angle determinations using 3-bond J-coupling (3JN-Hß, 3JCO-Hß, 3JHα-Hß) experiments. However, due to motional averaging, the assumption of a pure rotameric state can yield incorrect χ1 dihedral angles with incorrect stereospecific assignments. This was the case for three residues in the Pin1 WW domain (Lys28, Met30, and Asn44). Thus, stereoselective 1H,12C-labeling will be useful not only for NMR studies of large protein systems, but also for determining side chain rotamers and dynamics in any protein system.

Combining a PagP fusion protein system with nickel ion-catalyzed cleavage to produce intrinsically disordered proteins in E. coli.

Many proteins contain intrinsically disordered regions that are highly solvent-exposed and susceptible to post-translational modifications. Studying these protein segments is critical to understanding their physiologic regulation, but proteolytic degradation can make them difficult to express and purify. We have designed a new protein expression vector that fuses the target protein to the N-terminus of the integral membrane protein, PagP. The two proteins are connected by a short linker containing the sequence SRHW, previously shown to be optimal for nickel ion-catalyzed cleavage. The methodology is demonstrated for an intrinsically disordered segment of cardiac troponin I. cTnI[135-209]-SRHW-PagP-His6 fusion protein was overexpressed in Escherichia coli, accumulating in insoluble inclusion bodies. The protein was solubilized, purified using nickel affinity chromatography, and then cleaved with 0.5mM NiSO4 at pH 9.0 and 45 °C, all in 6M guanidine-HCl. Nickel ion-catalyzed peptide bond hydrolysis is an effective chemical cleavage technique under denaturing conditions that preclude the use of proteases. Moreover, nickel-catalyzed cleavage is more specific than the most commonly used agent, cyanogen bromide, which cleaves C-terminal to methionine residues. We were able to produce 15 mg of purified cTnI[135-209] from 1L of M9 minimal media using this protocol. The methodology is more generally applicable to the production of intrinsically disordered protein segments.

Metabolomic prediction of fetal congenital heart defect in the first trimester.

OBJECTIVE: The objective of the study was to identify metabolomic markers in maternal first-trimester serum for the detection of fetal congenital heart defects (CHDs). STUDY DESIGN: Mass spectrometry (direct injection/liquid chromatography and tandem mass spectrometry) and nuclear magnetic resonance spectrometry-based metabolomic analyses were performed between 11 weeks' and 13 weeks 6 days' gestation on maternal serum. A total of 27 CHD cases and 59 controls were compared. There were no known or suspected chromosomal or syndromic abnormalities indicated. RESULTS: A total of 174 metabolites were identified and quantified using the 2 analytical methods. There were 14 overlapping metabolites between platforms. We identified 123 metabolites that demonstrated significant differences on a univariate analysis in maternal first-trimester serum in CHD vs normal cases. There was a significant disturbance in acylcarnitine, sphingomyelin, and other metabolite levels in CHD pregnancies. Predictive algorithms were developed for CHD detection. High sensitivity (0.929; 95% confidence interval [CI], 0.92-1.00) and specificity (0.932; 95% CI, 0.78-1.00) for CHD detection were achieved (area under the curve, 0.992; 95% CI, 0.973-1.0). CONCLUSION: In the first such report, we demonstrated the feasibility of the use of metabolomic developing biomarkers for the first-trimester prediction of CHD. Abnormal lipid metabolism appeared to be a significant feature of CHD pregnancies.