From a human placental lambda gt11 cDNA library, we have isolated a cDNA clone that encodes the entire 215-residue amino acid sequence of HMG-1. Analysis of an internal sequence similarity suggests that the DNA-binding domains of HMG-1 are separated by a rather long and flexible linker segment. Southern blotting of DNA digested with BamHI indicated a highly variable number of genes (or pseudogenes) for HMG-1 in different species. Characterization of HMG-1 mRNA expression by Northern blotting showed that three mRNA species of approximately 1.0, 1.4 and 2.4 kb were expressed in all mammalian organs and cell lines examined. These included several rat organs at different stages of development. Northern analysis also suggested the occurrence of HMG-1 mRNA in an invertebrate and a plant species.
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 (T m ), 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 (⌬⌬G d 50°C ס −4 kcal/mole; ⌬T m ס −22°C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (⌬⌬G d 50°C ס −3 kcal/mole; ⌬T m ס −11°C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys 44 -Asp 45 peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (⌬⌬G d 50°C ס −1 kcal/mole; ⌬T m ס +2°C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (⌬⌬G d 50°C ס +1 kcal/mole; ⌬T m ס +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.Keywords: Disulfide-bond; cross-link; protein stability; differential scanning calorimetry; denaturation; folding Supplemental material: See www.proteinscience.org.The conformational stability of a protein is important to its function. Certain diseases such as Alzheimer's, prion, and cystic fibrosis, are associated with misfolded, unfolded, or aggregated proteins (Kelly 1996;Harper and Lansbury, Jr. 1997;Horwich and Weissman 1997, Qu et al. 1997). Much has been characterized regarding contributions to protein stability of hydrogen bonds, i...
The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.