Kandala V. R. Chary

MYCOBACTERIUM TUBERCULOSIS H37RV ESAT-6-CFP-10 COMPLEX FORMATION CONFERS THERMODYNAMIC AND BIOCHEMICAL STABILITY

The 6‐kDa early secretory antigenic target (ESAT‐6) and culture filtrate protein‐10 (CFP‐10), expressed from the region of deletion‐1 (RD1) of Mycobacterium tuberculosis H37Rv, are known to play a key role in virulence. In this study, we explored the thermodynamic and biochemical changes associated with the formation of the 1 : 1 heterodimeric complex between ESAT‐6 and CFP‐10. Using isothermal titration calorimetry (ITC), we precisely determined the association constant and free energy change for formation of the complex to be 2 × 107 m−1 and −9.95 kcal·mol−1, respectively. Strikingly, the thermal unfolding of the ESAT‐6–CFP‐10 heterodimeric complex was completely reversible, with a Tm of 53.4 °C and ΔH of 69 kcal·mol−1. Mixing of ESAT‐6 and CFP‐10 at any temperature below the Tm of the complex led to induction of helical conformation, suggesting molecular recognition between specific segments of unfolded ESAT‐6 and CFP‐10. Enhanced biochemical stability of the complex was indicated by protection of ESAT‐6 and an N‐terminal fragment of CFP‐10 from proteolysis with trypsin. However, the flexible C‐terminal of CFP‐10 in the complex, which has been shown to be responsible for binding to macrophages and monocytes, was cleaved by trypsin. In the presence of phospholipid membranes, ESAT‐6, but not CFP‐10 and the complex, showed an increase in α‐helical content and enhanced thermal stability. Overall, complex formation resulted in structural changes, enhanced thermodynamic and biochemical stability, and loss of binding to phospholipid membranes. These features of complex formation probably determine the physiological role of ESAT‐6, CFP‐10 and/or the complex in vivo. The ITC and thermal unfolding approach described in this study can readily be applied to characterization of the 11 other pairs of ESAT‐6 family proteins and for screening ESAT‐6 and CFP‐10 mutants.

Regulatory and Structural EF-Hand Motifs of Neuronal Calcium Sensor-1 : Mg2+ Modulates Ca2+ Binding, Ca2+-Induced Conformational Changes, and Equilibrium Unfolding Transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca(2+) signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca(2+)-specific) and structural (Ca(2+)- or Mg(2+)-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca(2+) binding using NMR and EF-hand mutants. Ca(2+) titration, as monitored by [(15)N,(1)H] heteronuclear single quantum coherence, suggests that Ca(2+) binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg(2+) binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca(2+)-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca(2+) and not Mg(2+). Ca(2+) binding induces conformational rearrangements in the protein by reversing Mg(2+)-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg(2+) with Ca(2+) reduces the Ca(2+)-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca(2+)-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg(2+)-bound NCS-1 are similar, the early unfolding transitions of Ca(2+)-bound NCS-1 are partially influenced in the presence of Mg(2+). This study demonstrates the importance of Mg(2+) as a modulator of calcium homeostasis and active-state behavior of NCS-1.

Solution Structure and Calcium-Binding Properties of M-Crystallin, A Primordial βγ-Crystallin from Archaea

The lens betagamma-crystallin superfamily has many diverse but topologically related members belonging to various taxa. Based on structural topology, these proteins are considered to be evolutionarily related to lens crystallins, suggesting their origin from a common ancestor. Proteins with betagamma-crystallin domains, although found in some eukaryotes and eubacteria, have not yet been reported in archaea. Sequence searches in the genome of the archaebacterium Methanosarcina acetivorans revealed the presence of a protein annotated as a betagamma-crystallin family protein, named M-crystallin. Solution structure of this protein indicates a typical betagamma-crystallin fold with a paired Greek-key motif. Among the known structures of betagamma-crystallin members, M-crystallin was found to be structurally similar to the vertebrate lens betagamma-crystallins. The Ca(2+)-binding properties of this primordial protein are somewhat more similar to those of vertebrate betagamma-crystallins than to those of bacterial homologues. These observations, taken together, suggest that amphibian and vertebrate betagamma-crystallin domains are evolutionarily more related to archaeal homologues than to bacterial homologues. Additionally, identification of a betagamma-crystallin homologue in archaea allows us to demonstrate the presence of this domain in all the three domains of life.

Solution structure of d-GAATTCGAATTC by 2D NMR: A new approach to determination of sugar geometries in DNA segments

A new approach based on the correlated spectroscopy (COSY) in 2D NMR has been described for determination of sugar geometries in oligonucleotides. Under the usual low resolution conditions employed in COSY, the intensities of cross peaks depend on the magnitudes of coupling constants. There are five vicinal coupling constants in a deoxyribose ring which are sensitive to the sugar geometry. The presence, absence and rough comparison of relative intensities of COSY cross peaks arising from such coupling constants enable one to fix the sugar conformation to a fair degree of precision. The methodology has been applied to d‐GAATTCGAATTC. It is observed that ten out of the twelve nucleotide units in this sequence exhibit a rare Ol′‐endo geometry. The EcoRI cleavage sites (between G and A) in the dodecanucleotide show an interesting variation in the conformation with the two sugars attached to the Gs acquiring a geometry between C2′‐endo and C4′‐endo.

Copper Alters Aggregation Behavior of Prion Protein and Induces Novel Interactions between Its N- and C-terminal Regions

Background: Role of copper as an attenuator or facilitator in prion diseases is controversial. Results: Copper-bound PrP does not aggregate at physiological temperature and shows two novel interactions between its N- and C-terminal domains. Conclusion: Copper may act as an attenuator in prion diseases and induces novel long range inter-domain interactions in PrP. Significance: This study might help in understanding the role of copper in prion diseases. Copper is reported to promote and prevent aggregation of prion protein. Conformational and functional consequences of Cu2+-binding to prion protein (PrP) are not well understood largely because most of the Cu2+-binding studies have been performed on fragments and truncated variants of the prion protein. In this context, we set out to investigate the conformational consequences of Cu2+-binding to full-length prion protein (PrP) by isothermal calorimetry, NMR, and small angle x-ray scattering. In this study, we report altered aggregation behavior of full-length PrP upon binding to Cu2+. At physiological temperature, Cu2+ did not promote aggregation suggesting that Cu2+ may not play a role in the aggregation of PrP at physiological temperature (37 °C). However, Cu2+-bound PrP aggregated at lower temperatures. This temperature-dependent process is reversible. Our results show two novel intra-protein interactions upon Cu2+-binding. The N-terminal region (residues 90–120 that contain the site His-96/His-111) becomes proximal to helix-1 (residues 144–147) and its nearby loop region (residues 139–143), which may be important in preventing amyloid fibril formation in the presence of Cu2+. In addition, we observed another novel interaction between the N-terminal region comprising the octapeptide repeats (residues 60–91) and helix-2 (residues 174–185) of PrP. Small angle x-ray scattering studies of full-length PrP show significant compactness upon Cu2+-binding. Our results demonstrate novel long range inter-domain interactions of the N- and C-terminal regions of PrP upon Cu2+-binding, which might have physiological significance.

J-scaling in two-dimensional homonuclear correlated spectroscopy for enhancement of cross peak intensities

New pulse schemes have been proposed for enhancement of cross peak intensities in two-dimensional homonuclear correlated spectra. These rely on non-selective scaling up of spin-spin coupling constant values and refocusing of the multiplet components. Experimental spectra illustrating the application of the new pulse schemes are presented.

J tuning of SUPERCOSY as an aid to resonance assignments

Nuclear magnetic resonance spectroscopy has been extensively used for conformational analysis of biological molecules (I). With the advent of two-dimensional NMR techniques, it has become possible to obtain individual resonance assignments and detailed structural information on large molecules having molecular weights of the order of 6000-7000 (2-15). Recently a pulse sequence has been proposed (26) which is superior to the normal COSY (I 7, 18) scheme for observation of coupling correlations in complicated spin systems. The new pulse scheme has the sequence: 90-t,-A-180-A-90-A-180-At2, where A is a fixed delay and t, and t2, are, respectively, the usual evolution and detection periods of two-dimensional spectroscopy. The value of A depends upon the nature of the spin systems and the magnitudes of the coupling constants (J) between the spins. For an AX spin system, the optimum value of A is 1/4J and for an AX2 system, it is 1/2J. The main advantage of SUPERCOSY over the COSY scheme lies in the fact that the cross-peaks and the diagonal peaks in SUPERCOSY consist of in-phase and antiphase components respectively, while exactly the opposite is true in the COSY spectrum. Consequently, under conditions of overlap or poor digital resolution, cross-peaks build much faster in intensity, while the diagonal peaks tend to grow relatively slower. The fact that the fixed delay A in the SUPERCOSY pulse scheme depends on the value of J enables one to tune the experiment to a particular value of J which may be of special interest. In other words, cross-peaks arising from particular values of J can be selectively enhanced. We have used this principle here to observe selective proton couplings so as to arrive at a unique resonance assignment of the ring protons in the tryptophan residue of a decapeptide, leutinizing hormone releasing hormone (LHRH). Figure 1 shows SUPERCOSY spectra for two different values of A, 30 ms (A) and 40 ms (B). While the complete analysis will be published separately, the intention of this note is to demonstrate how J tuning of SUPERCOSY can be used as an aid in resonance assignment procedures. The two spectra shown in Fig. 1 depict variations in the intensities of several cross-peaks and diagonal peaks. Figure 2 shows expansions of the aromatic regions of the spectra (02 = 6.4-l 1 .O ppm; WI = 6.4-l 1 .O ppm). The peaks labeled A, B, C, D, etc, identify the J-coupling correlations between various ring protons. For example, in the spectrum of Fig. 2A,

Identification of C-terminal neighbours of amino acid residues without an aliphatic 13Cγ as an aid to NMR assignments in proteins

We propose a methodology that uses GFT (3,2)D CB(CACO)NNH experiment to rapidly collect the data and readily identify six amino acid residue types (Ala, Asn, Asp, Cys, Gly and Ser) in any given protein. Further, the experiment can distinguish the redox state of Cys residues. The proposed experiment in its two forms will have wide range of applications in resonance assignment strategies and structure determination of proteins.

Equilibrium Unfolding of Neuronal Calcium Sensor-1 N-TERMINAL MYRISTOYLATION INFLUENCES UNFOLDING AND REDUCES PROTEIN STIFFENING IN THE PRESENCE OF CALCIUM

Neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein of the calcium sensor family, modulates various functions in intracellular signaling pathways. The N-terminal glycine in this protein is myristoylated, which is presumably necessary for its physiological functions. In order to understand the structural role of myristoylation and calcium on conformational stability, we have investigated the equilibrium unfolding and refolding of myristoylated and non-myristoylated NCS-1. The unfolding of these two forms of NCS-1 in the presence of calcium is best characterized by a five-state equilibrium model, and multiple intermediates accumulate during unfolding. Calcium exerts an extrinsic stabilizing effect on both forms of the protein. In the absence of calcium, the stability of both forms is dramatically decreased, and the unfolding follows a four-state equilibrium model. The equilibrium transitions are fully reversible in the presence of calcium. Myristoylation affects the pattern of equilibrium transitions substantially but not the number of intermediates, suggesting a structural role. Our data suggest that myristoylation reduces the stiffening of the protein during initial unfolding in the presence of calcium. The effects of myristoylation are more pronounced when calcium is present, suggesting a relationship between them. Inactivating the third EF-hand motif (E120Q mutant) drastically affects the equilibrium unfolding transitions, and calcium has no effect on these transitions of the mutants. The unfolding transitions of both forms of the mutant are similar to the transitions followed by the apo forms of myristoylated and non-myristoylated NCS-1. These results suggest that the role of myristoylation in unfolding/refolding of the protein is largely dependent on the presence of calcium.

A Natively Unfolded βγ-Crystallin Domain from Hahella chejuensis

To date, very few βγ-crystallins have been identified and structurally characterized. Several of them have been shown to bind Ca(2+) and thereby enhance their stability without any significant change in structure. Although Ca(2+)-induced conformational changes have been reported in two putative βγ-crystallins from Caulobacter crescentus and Yersinia pestis, they are shown to be partially unstructured, and whether they acquire a βγ-crystallin fold is not known. We describe here a βγ-crystallin domain, hahellin, its Ca(2+) binding properties and NMR structure. Unlike any other βγ-crystallin, hahellin is characterized as a pre-molten globule (PMG) type of natively unfolded protein domain. It undergoes drastic conformational change and acquires a typical βγ-crystallin fold upon Ca(2+) binding and hence acts as a Ca(2+)-regulated conformational switch. However, it does not bind Mg(2+). The intrinsically disordered Ca(2+)-free state and the close structural similarity of Ca(2+)-bound hahellin to a microbial βγ-crystallin homologue, Protein S, which shows Ca(2+)-dependent stress response, make it a potential candidate for the cellular functions. This study indicates the presence of a new class of natively unfolded βγ-crystallins and therefore the commencement of the possible functional roles of such proteins in this superfamily.