Ramaswamy Krishnamoorthi

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V.

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross‐links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross‐links, protein flexibility in the vicinity of a cross‐link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor‐V (CMTI‐V; 7 kD) by differential scanning calorimetry. CMTI‐V offers the advantage of a large, flexible, and solvent‐exposed loop not involved in extensive intra‐molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (Tm), an indicator of native state stabilization, for CMTI‐V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide‐removed C3S/C48S (ΔΔGd50°C = −4 kcal/mole; ΔTm = −22°C), the native state is destabilized more than the denatured state; this also applies to the less‐stable CMTI‐V* (ΔΔGd50°C = −3 kcal/mole; ΔTm = −11°C), in which the disulfide‐containing loop is opened by specific hydrolysis of the Lys44‐Asp45 peptide bond; (2) In the less stable, disulfide‐inserted E38C/W54C (ΔΔGd50°C = −1 kcal/mole; ΔTm = +2°C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide‐engineered V42C/R52C (ΔΔGd50°C = +1 kcal/mole; ΔTm = +17°C), the native state is more stabilized than the denatured state. These results show that a cross‐link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI‐V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross‐link on the denatured state of CMTI‐V was estimated directly by means of a four‐state thermodynamic cycle consisting of native and denatured states of CMTI‐V and CMTI‐V*. Overall, the results show that an enthalpy‐entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross‐link, and residual structure in the denatured state.

A new protein inhibitor of trypsin and activated hageman factor from pumpkin (Cucurbita maxima) seeds

A protein inhibitor (CMTI‐V; M r 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin‐affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 × 10−8 and 4.1 × 10−8 M, respectively. The primary structure of CMTI‐V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N‐acetylates Ser. The reactive site has been established to be the peptide bond between Lys44‐Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.

Solution conformations of proline rings in proteins studied by NMR spectroscopy

SummaryThree different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine β-, γ- and δ-hydrogens have been made on the basis of 1H-1H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs α-β3, β3-γ3, β2-γ2, γ2-δ2, and γ3-δ3 (2.3 Å) are shorter than those in the pairs α-β2, β2-γ3, β3-γ2, γ2-δ3, and γ3-δ2 (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the β3-γ2 and γ2-δ3 spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the α-β2, β2-γ3, and γ3-δ2 vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.

Natural-abundance 15N NMR studies of turkey ovomucoid third domain. Assignment of peptide 15N resonances to the residues at the reactive site region via proton-detected multiple-quantum coherence

Abstract Heteronuclear two-dimensional 1 H{ 15 N} multiple-quantum (MQ) spectroscopy has been applied to a protein sample at natural abundance: ovomucoid third domain from turkey ( Meleagris gallopavo ), a serine proteinase inhibitor of 56 amino acid residues. Peptide amide 1 H NMR assignments obtained by two-dimensional 1 H{ 1 H} NMR methods (R. Krishnamoorthi and J. L. Markley, unpublished data) led to identification of the corresponding 1 H{ 15 N} MQ coherence cross peaks. From these, 15 N NMR chemical shifts were determined for several specific backbone amide groups of amino acid residues located around the reactive site region of the inhibitor. The results suggest that amide 15 N chemical shifts, which are readily obtained in this way, may serve as sensitive probes for conformational studies of proteins.

Solvent isotope effects on NMR spectral parameters in high-spin ferric hemoproteins: an indirect probe for distal hydrogen bonding.

The influence of solvent isotope composition on 1H-NMR resonance position and linewidth of heme methyls has been investigated for a variety of high-spin ferric hemoproteins for the purpose of detecting hydrogen-bonding interactions in the heme cavity. Consistently larger hyperfine shifts and paramagnetic linewidths in 2H2O than 1H2O are observed for metmyoglobins and methemoglobin possessing a coordinated water molecule. The analysis of the dynamics of labile proton exchange in sperm whale metmyoglobin, and the absence of any isotope effects in the five-coordinate Aplysia metmyoglobin, indicate that the significant axial modulation of heme electronic structure by solvent isotope is consistent with arising from distal hydrogen-bonding interactions. The presence or absence of similarly large isotope effects on shifts and linewidths in other hemoproteins, depending on the presence of a bound water in the distal heme pocket, suggests that this isotope effect can serve as a probe for the presence of such bound water. The absence of any detectable isotope effect on either shifts or linewidths in resting-state horseradish peroxidase supports a five-coordinate structure with bound water absent from the vicinity of the iron.

Self-association of an insect β-1,3-glucan recognition protein upon binding laminarin stimulates prophenoloxidase activation as an innate immune response

Background: The N-terminal domain of an insect β-glucan recognition protein (N-βGRP) stimulates innate immune responses. Results: N-βGRP forms soluble and insoluble protein-carbohydrate complexes involving specific protein-protein interactions, which activates the prophenoloxidase pathway. Conclusion: Assembly of βGRP oligomers stimulated by binding to microbial polysaccharide triggers phenoloxidase activation. Significance: βGRP interactions stimulate an innate immune response by a novel mechanism. Insect β-glucan recognition protein (βGRP), a pathogen recognition receptor for innate immune responses, detects β-1,3-glucan on fungal surfaces via its N-terminal carbohydrate-binding domain (N-βGRP) and triggers serine protease cascades for the activation of prophenoloxidase (pro-PO) or Toll pathways. Using biophysical and biochemical methods, we characterized the interaction of the N-terminal domain from Manduca sexta βGRP2 (N-βGRP2) with laminarin, a soluble form of β-1,3-glucan. We found that carbohydrate binding by N-βGRP2 induces the formation of two types of protein-carbohydrate complexes, depending on the molar ratio of carbohydrate to protein ([C]/[P]). Precipitation, analytical ultracentrifugation, and chemical cross-linking experiments have shown that an insoluble aggregate forms when the molar ratio of carbohydrate to protein is low ([C]/[P] ∼ 1). In contrast, a soluble complex, containing at least five N-βGRP2 molecules forms at a higher molar ratio of carbohydrate/protein ([C]/[P] >5). A hypothesis that this complex is assembled partly due to protein-protein interactions was supported by chemical cross-linking experiments combined with LC-MS/MS spectrometry analysis, which permitted identification of a specific intermolecular cross-link site between N-βGRP molecules in the soluble complex. The pro-PO activation in naive plasma was strongly stimulated by addition of the insoluble aggregates of N-βGRP2. The soluble complex with laminarin formed in the plasma also stimulated pro-PO activation, but at a lower level. Taken together, these results provide experimental evidence for novel mechanisms in which associations of βGRP with microbial polysaccharide promotes assembly of βGRP oligomers, which may form a platform needed to trigger the pro-PO pathway activation cascade.

An initial event in the insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein.

In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the nuclear magnetic resonance (NMR) solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone (15)N-(1)H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-βGRP-laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-βGRP-laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of an X-ray crystallographic structure of the N-βGRP-laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements conducted with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of the N-βGRP-laminarin macro complex and that a decreased stability is accompanied by a reduced level of activation of the proPO pathway. An increased level of β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of the βGRP-β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway.

Expression, refolding, and activation of the catalytic domain of human blood coagulation factor XII.

Human blood coagulation factor XII (FXII; 80 kDa) contains a C-terminal serine protease zymogen domain, which becomes activated upon contacting a negative surface. Activated FXII (alphaFXIIa) brings about reciprocal activation of FXII and kallikrein that by further hydrolysis produces the free catalytic domain (betaFXIIa; 28 kDa). Increased levels of alphaFXIIa are associated with coronary heart disease, sepsis, and diabetes. Biophysical investigation of the structural basis of activation, substrate specificity, and regulation of FXII requires an efficient bacterial system for producing the wild-type and mutant recombinant proteins. Here, the cDNA of the zymogen domain of FXII (betaFXII) was cloned into the pET-28a(+) vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) and overexpressed. The multi-disulfide, recombinant protein, His(6)-betaFXII (rbetaFXII), expressed as an inclusion body, was purified by means of a Ni(2+)-charged resin. The matrix-bound rbetaFXII was subjected to refolding with the glutathione redox system and activated by the in vivo activator, kallikrein. The active form, rbetaFXIIa, obtained in milligram quantities, exhibited similar structural and comparable functional properties relative to human betaFXIIa, as indicated by circular dichroism spectroscopy and kinetics of substrate hydrolysis. Thermodynamics of enzyme:inhibitor complex formation, including the expected 1:1 stoichiometry, was determined for rbetaFXIIa by isothermal calorimetric titration with a specific recombinant protein inhibitor, Cucurbita maxima trypsin inhibitor-V (rCMTI-V; 7kDa).

Proton magnetic resonance study of the influence of heme 2,4 substituents on the exchange rates of labile protons in the heme pocket of myoglobin

Four exchangeable protons with large hyperfine shifts are assigned in the heme pocket of sperm whale met-cyano myoglobin reconstituted with heme possessing acetyl groups, ethyl groups, bromines, and hydrogens at the 2,4 position, using both relaxation and chemical-shift data. The four protons arise from the ring NHs of the proximal (F8), distal (E7), and FG2 histidines, and the peptide NH of His F8. The similarity of all chemical shifts to those of the native protein as well as the invariance of the relaxation rates of the distal histidyl ring NH dictate essentially the same structure for the heme cavity of both native and reconstituted proteins. The exchange rates with bulk water of the four labile proteins in each modified protein were determined by saturation-transfer and line width methods. All four labile protons were found to have the same exchange rate as in the native protein for acetyl and ethyl 2,4 substituents; the two resolved labile protons in the derivative with 2,4 bromine were also unchanged. The reconstituted protein with hydrogens at the 2,4 position exhibited slower exchange rates for three of the four protons, indicating an increased dynamic stability of the heme pocket in the absence of bulky 2,4 substituents.

Chemical warfare agent simulants in Gamble’s fluid: Is the fluid toxic? Can it be made safer by inclusion of solid nanocrystalline metal oxides?

The reactions of chemical warfare agent simulants, 2-chloroethyl ethyl sulfide (2-CEES) and di-i-propyl fluoro phosphate (DFP), in fluids have been investigated. Data analyses confirm the major degradation pathway to be hydrolysis of 2-CEES to 2-hydroxyethyl ethyl sulfide, along with minor self-condensation products. Among the three fluids examined, 2-CEES degradation was the fastest in Gamble’s fluid during a 96 h period. Upon addition of Exceptional Hazard Attenuation Materials (EHAMs) to 2-CEES containing Gamble’s fluid, degradation was generally improved during the first 24 h period. The 96 h outcome was similar for fluid samples with or without EHAM 2 and EHAM 4. EHAM 1-added fluid contained only one degradation product, 2-nitroethyl ethyl sulfide. DFP degradation was the slowest in Gamble’s fluid, but was enhanced by the addition of EHAMs. FTIR and solid state 31P NMR confirm the destructive adsorption of 2-CEES and DFP by the EHAMs. The results collectively demonstrate that 2-CEES and DFP decompose to various extents in Gamble’s fluid over a 96 h period but the fluid still contains a considerable amount of intact simulant. EHAM 1 appears to be promising for 2-CEES and DFP mitigation while EHAM 2 and EHAM 4 work well for early on concentration reduction of 2-CEES and DFP.