Biomedical subjects

Ranajeet Ghose

Publications and source records attributed to Ranajeet Ghose.

9 recordsLinked to original sources

PubMed2006

Solution structure and dynamics of the N-terminal cytosolic domain of rhomboid intramembrane protease from Pseudomonas aeruginosa: insights into a functional role in intramembrane proteolysis.

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel alpha/beta fold and represents the first detailed structural insight into this class of intramembrane proteases. We find evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds-milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.

Binding Sites↗

PubMed2006

Structure and dynamics of ASC2, a pyrin domain-only protein that regulates inflammatory signaling.

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-kappaB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha2-alpha3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha2-alpha3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha2-alpha3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.

Amino Acid Sequence↗

PubMed2006

The pH-dependence of amide chemical shift of Asp/Glu reflects its pKa in intrinsically disordered proteins with only local interactions.

Detailed knowledge of the pH-dependence of ionizable residues in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein folding and stability. The reassembly of E. coli Thioredoxin (Trx) by complementation of its two disordered fragments (1-37/38-108) provides a folded heterodimer in equilibrium with its unfolded state which, based on circular dichroism and NMR spectroscopy, consists of two unfolded monomers. To gain insight into the role of electrostatics in protein folding and stability, we compared the pH-dependence of the carboxylate sidechain chemical shift of each Asp/Glu against that of its backbone amide chemical shift in the unfolded heterodimer. We monitored via C(CO)NH experiments four Asp and four Glu in fragments 38 to 108 (C37) of Trx in the pH range from 2.0 to 7.0 and compared them with results from (1)H(15)N HSQC experiments [Pujato et al., Biophys. J., 89 (2005) 3293-3302]. The (1)H(15)N HSQC analysis indicates two segments with quite distinct behavior: (A) a segment from Ala57 to Ala108 in which ionizable residues have up to three contiguous neighbors with pH-dependent backbone amide shifts, and (B) a segment of fifteen contiguous pH-dependent backbone amide shifts (Leu42 to Val56) in which two Asp and two Glu are implicated in medium range interactions. In all cases, the titration curves are simple modified sigmoidals from which a pH-midpoint (pH(m)) can be obtained by fitting. In segment A, the pH(m) of a given backbone amide of Asp/Glu mirrors within 0.15 pH-units that of its carboxylate sidechain (i.e., the pK(a)). In contrast, segment B shows significant differences with absolute values of 0.46 and 0.74 pH-units for Asp and Glu, respectively. The dispersion in the pH(m) of the backbone amide of Asp/Glu is also different in the two segments. Segment A shows a dispersion of 0.31 and 0.17 pH-units for Asp and Glu, respectively. Segment B shows a substantially larger dispersion (0.50 and 1.08 pH-units for Asp and Glu, respectively). In both segments, the dispersion in the pH(m) of its backbone amide is larger than in the pK(a) of the carboxylate sidechain (the latter is only 0.17 and 0.52 pH-units for Asp and Glu, respectively). Our results indicate that the pH(m) of the backbone amide chemical shift of Asp/Glu in a disordered polypeptide segment is a good predictor of its pK(a) whenever there are none or few neighboring backbone amides with similar pH-dependence.

Amides↗

PubMed2006

Detection of correlated dynamics on multiple timescales by measurement of the differential relaxation of zero- and double-quantum coherences involving sidechain methyl groups in proteins.

Multiple effects may lead to significant differences between the relaxation rates of zero-quantum coherences (ZQC) and double-quantum coherences (DQC) generated between a pair of nuclei in solution. These include the interference between the anisotropic chemical shifts of the two nuclei participating in formation of the ZQC or DQC, the individual dipolar interactions of each of the two nuclei with the same proton, and the slow modulation of the isotropic chemical shifts of the two nuclei due to conformational exchange. Motional events that occur on a timescale much faster than the rotational correlation time (ps-ns) influence the first two effects, while the third results from processes that occur on a far slower timescale (mus-ms). An analysis of the differential relaxation of ZQC and DQC is thus informative about dynamics on the fast as well as the slow timescales. We present here an experiment that probes the differential relaxation of ZQC and DQC involving methyl groups in protein sidechains as an extension to our recently proposed experiments for the protein backbone. We have applied the methodology to (15)N, (13)C-labeled ubiquitin and used a detailed analysis of the measured relaxation rates using a simple single-axis diffusion model to probe the motional restriction of the C(next)H(next) bond vector where C(next) is the carbon that is directly bonded to a sidechain methyl carbon (C(methyl)). Comparison of the present results with the motional restriction of the C(next)C(methyl) bond (S(axis)(2)) reveals that the single-axis diffusion model, while valid in the fringes of the protein and for shorter chain amino acids, proves inadequate in the central protein core for long chain, asymmetrically branched amino acids where more complex motional models are necessary, as is the inclusion of the possibility of correlation between multiple motional modes. In addition, the present measurements report on the modulation of isotropic chemical shifts due to motion on the mus-ms timescale. Three Leu residues (8, 50, and 56) are found to display these effects. These residues lie in regions where chemical shift modulation had been detected previously both in the backbone and sidechain regions of ubiquitin.

Anisotropy↗

PubMed2006

NMR determination that an extended BH3 motif of pro-apoptotic BID is specifically bound to BCL-XL.

The BH3 motif of the pro-survival family of proteins, BCL, is also present in pro-apoptotic proteins like BID and BAX. Homo- and hetero-oligomerization interactions of the BH3 motif are generally recognized as the critical component of their apoptotic activities. In full-length BID, the putative hydrophobic binding surface of its BH3 motif is substantially occluded by intramolecular contacts, many of which are removed on BID's transformation to tBID by cleavage with caspase 8, required for tBID's pro-apoptotic action on mitochondria, thereby releasing cytochrome c.

Amino Acid Motifs↗

PubMed2005

Voltage gating and anions, especially phosphate: a model system.

The voltage sensor of voltage gated sodium and potassium channels consists of four sets of transmembrane segments, of which one, called S4, contains at least four arginines; these are presumed to each carry positive charges. The channel opening is usually attributed to the outward (i.e., toward the extracellular side of the membrane) motion of S4. The evidence for this motion is based on certain experiments that appear to show differential access to parts of S4 from the intracellular and extracellular sides of the membrane in the open and closed states. A newly available structure [S.B. Long, E.B. Campbell and R. MacKinnon, Crystal structure of a mammalian voltage-dependent Shaker family K(+) channel. Science 309 (2005) 897-903; S.B. Long, E.B. Campbell, R. MacKinnon, Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309 (2005) 903-908][1,2] has now been used to argue for a large scale motion, although, as a static structure, it is not conclusive. In this paper, we consider the effect of anions in the surrounding medium. Phosphate is present in the intracellular as well as the extracellular fluid, apparently at hundreds of micromolar concentration, or more. There is evidence in the literature suggesting that phosphate-arginine complexes are rather strong. In a recent calculation one of us [M.E. Green, A possible role for phosphate in complexing the arginines of S4 in voltage gated channels J. Theor. Biol. 233 (2005) 337-341][3] has shown that a model peptide with a 2:1 arg:phosphate complex should have a favorable geometry. Here, we present NMR evidence of the existence of phosphate complexes of a model peptide with two arginines separated by two hydrophobic residues, the same spacing as in S4 segments. The complexes (there are different complexes for HPO(4)(2-) and for H(2)PO(4)(-) [3]) form with concentrations of peptide in the range of hundreds of micromolar, making it significant in the biological context. NMR spectra provide changes in chemical shift as functions of both phosphate concentration and pH. The resulting curves show titration of the phosphate, with its standard pK. Possible implications for other anion-S4 interactions, including ion pairs rather than complexes, as with Cl(-), are also discussed.

Animals↗

PubMed2004

Dynamic influences on a high-affinity, high-specificity interaction involving the C-terminal SH3 domain of p67phox.

An analysis of the backbone dynamics of the C-terminal Src homology 3 (SH3) domain of p67(phox), p67(phox)SH3(C), in complex with a 32-residue high-affinity (K(d) = 24 nM) peptide, Pf, from the C-terminal region of p47(phox) is presented. This paper represents the first detailed analysis of the backbone dynamics and the ligand-induced changes therein of a high-affinity, high-specificity interaction involving an SH3 domain. The dynamic features are compared with those in the high-affinity, highly specific interaction between the SH3 domain of C-terminal Src kinase (Csk-SH3) and a proline-rich peptide from proline-enriched phosphatase (PEP). Both systems share common dynamic features especially in the canonical PxxP motif recognition surface where slow micro- to millisecond time scale dynamics persist on complex formation especially in several residues that are implicated in ligand recognition and in stabilizing the SH3 fold. These residues are highly conserved in SH3 domains. Ile505, which lies outside the PxxP recognition motif on p67(phox)SH3(C) and is key in conferring high specificity to the p67(phox)SH3(C)/Pf interaction, becomes more disordered upon complex formation. This behavior is similar to that seen in the residues that constitute the specificity surface in Csk-SH3.

Amino Acids↗

PubMed2004

Probing slow backbone dynamics in proteins using TROSY-based experiments to detect cross-correlated time-modulation of isotropic chemical shifts.

The difference in the relaxation rates of zero-quantum (ZQ) and double-quantum (DQ) coherences is the result of three principal mechanisms. These include the cross-correlation between the chemical shift anisotropies of the two participating nuclei, dipolar interactions with remote protons as well as interference effects due to the time-modulation of their isotropic chemical shifts as a consequence of slow micros-ms dynamics. The last effect when present, dominates the others resulting in large differences between the relaxation rates of ZQ and DQ coherences. We present here four sets of TROSY-based (Salzmann et al., 1998) experiments that measure this effect for several pairs of backbone nuclei including (15)N, (13)C(alpha) and (13)C'. These experiments allow the detection of the presence of slow dynamic processes in the protein backbone including correlated motion over two and three bonds. Further, we define a new parameter chi which represents the extent of correlated motion on the slow (micros-ms) timescale. This methodology has been applied to (15)N,(13)C,REDPRO-(2)H-labeled (Shekhtman et al., 2002) human ubiquitin. The ubiquitin backbone is seen to exhibit extensive dynamics on the slow timescale. This is most pronounced in several residues in the N-terminal region of the alpha-helix and in the loop connecting the strands beta(4) and beta(5). These residues which include Glu24, Asn25, Glu51 and Asp 52 form a continuous surface. As an additional benefit, the measured rates confirm the dependence of the (13)C(alpha) chemical shift tensor on local secondary structure of the protein backbone.

Humans↗

PubMed2002

NMR structure determination and investigation using a reduced proton (REDPRO) labeling strategy for proteins.

We present here a stable isotope labeling technique for proteins, which seeks the appropriate compromise between the advantages of (a) random isotope labeling, with its large number of protons available for structure determination, and (b) selective labeling to generate isolated proton spins decreasing spectral complexity and improving relaxation properties of NMR experiments. The described reduced proton (REDPRO) procedure results in side-chain specific protonation of overexpressed proteins, which is highly selective. The REDPRO labeling scheme provides a sufficient number of NOE constraints for structure calculation. Dramatically improved relaxation properties of the heteronuclear magnetization transfer coupled with TROSY advantages make the proposed labeling scheme an attractive approach for study of high molecular weight protein targets, their ligand sites, and interdomain interactions.

Magnetic Resonance Spectroscopy↗