Acid denaturation

Lashuel HA, Lai Z, Kelly JW. Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation implications for wild-type, V30M, and L55P amyloid fibril formation. Biochemistry 1998 37 17851-17864. 

Table V shows the results of this analysis for the Pn-helix fraction of several proteins denatured by heat, cold, acid, and Gdm HCl/urea. There is rather good consistency among the estimated Pn-helix contents for proteins denatured by a given agent, except for acid-denatured proteins, which show more variability. The chemically denatured proteins have 30 5% Pn-helix content near 0°C. At the other extreme, heat-denatured proteins have Pn-helix contents near 0%, with lysozyme having the highest value (8%). Although there are only two examples of cold-denatured proteins in Table V,2 they both have Pn-helix contents of about 20%. Acid-denatured proteins have Pn-helix contents ranging from 0 to 16%.

Proteins unfolded by GdmHCl or urea will have a dominant conformation, Pn- At low temperatures we find about one-third of the residues in chemically denatured proteins in the Pn-helix conformation, with two-thirds in the form of the high-temperature ensemble. Since at least one-third of the residues in this ensemble are isolated Pn residues or in Pn helices of two or three residues, the total Pn content will be 50% or greater. The Pn content of cold- and acid-denatured proteins will be substantial, probably >40%, but not as large as in chemically denatured proteins. 

The near-UV CD spectrum of carbonic anhydrase (Fig. 39) is rather strong and displays substantial fine structure at pH 7 (Wong and Hamlin, 1974). In the molten globule and the acid-denatured forms, the near-UV CD spectrum is nearly abolished, although the authors report weak residual positive CD through the aromatic region. 

Fig. 39. Near-UV CD spectrum of bovine carbonic anhydrase B. Native state, pH 7 (—) acid-denatured state, pH 2 (---). See note in legend to Fig. 38. From Wong and Hamlin (1974). Biochemistry 13, 2678-2683, with permission. 1974, American Chemical Society.

Zerovnik, E., Jerala, R., Kroon-Zitko, L., Turk, V., and Lohner, K. (1997). Characterization of the equilibrium intermediates in acid denaturation of human stefin B. Eur.J. Biochem. 245, 364-372. 

Matsuura, J.E., A.E. Morris, R.R. Ketchem, E.H. Braswell, R. Klinke, W.R. Gom-botz, and R.L. Remmele, Jr. 2001. Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer evidence of a reversible acid-denatured molten globule. Arch Biochem Biophys 392 208-218. 

Figure 7.1 (a) The denatured conformation of the zinc metalloenzyme carbonic anhydrase and the ESI mass spectra obtained under acidic denaturing conditions, (b) The ESI mass spectra obtained under native-state conditions. The decon-voluted ESI mass spectra of carbonic anhydrase reveals the protein molecular weight. The three dimensional structure is protein Data Bank ID IBNl. 

One type of the constituent metallocenters in the MoFe protein has the properties of a somewhat independent structural entity. This component, referred to as the FeMo cofactor (FeMo-co), was first identified by Shah and Brill (1977) as the stable metallocluster extracted from acid-denatured MoFe protein. The FeMo-co was able to fully activate a defective protein in the extracts of mutant strain UW45, a protein which subsequently was shown to contain the P clusters but not the EPR-active center. The isolated cofactor accounted for the total S = t system observed by EPR and Mdssbauer spectroscopies of the holo-MoFe protein (Rawlings et al., 1978). Elemental analysis indicated a composition of Mo Fee-8 Se-g for the cofactor, which, if there are two FeMo-co s per a2 2> accounts for all the molybdenum and approximately half the iron in active enzyme (Nelson etai, 1983). Although FeMo-co has been extensively studied [reviewed in Burgess (1990)] the structure remains enigmatic. To date, all attempts to crystallize the cofactor have failed. This is possibly due to the instability and resultant heterogeneity of the cofactor when removed from the protein. Also, there is a paucity of appropriate models for spectral comparison (see Coucouvanis, 1991, for a recent discussion). Final resolution of this elusive structure may require its determination as a component of the holoprotein. 

In the stomach, hydrochloric acid denatures dietary proteins, making them more susceptible to proteases. Pepsin, an enzyme secreted in zymogen form by the serous cells of the stomach, releases peptides and a few free amino acids from dietary proteins. 

Doi, E. and Jirgensons, B. 1970. Circular dichroism studies on the acid denaturation of y-immunoglobulin G and its fragments. Biochemistry 9, 1066-1073. 

Chemical studies also support the indicated mechanism. For example, the P-oxoacid intermediate formed in step b of Eq. 13-48 or Fig. 13-12 has been identified as a product released from the enzyme by acid denaturation during steady-state turnover.273 274 Isotopic exchange with 3H in the solvent275 and measurement of 13C isotope effects277 have provided additional verification of the mechanism. The catalytic activity of the enzyme is determined by ionizable groups with pKa values of 7.1 and 8.3 in the ES complex.278... 

Nitrogenase, which catalyzes the reduction of N2 to two molecules of NH3, has a different molybdenum -iron cofactor (FeMo-co). It can be obtained by acid denaturation of the very oxygen-labile iron-molybdenum protein of nitrogenase followed by extraction with d i methyl formamide.655,656 The coenzyme is a complex Fe-S-Mo cluster also containing homocitrate with a composition MoFe7S9-homocitrate (see Fig. 24-3). Nitrogenase and this coenzyme are considered further in Chapter 24. 

Each P-cluster is actually a joined pair of cubane-type clusters, one Fe4S4 and one Fe4S3 with two bridging cysteine -SH groups and one iron atom bonded to three sulfide sulfur atoms (Fig. 24-3).17/23 The FeMo-coenzyme can be released from the MoFe-protein by acid denaturation followed by extraction with dimethylformamide.24 While homocitrate was identified as a component of the isolated coenzyme, the three-dimensional structure of FeMo-co was deduced from X-ray crystallography of the intact molybdenum-iron protein.14/17/18... 

General details about denatured states were discussed in Chapter 17, section B. Specific details are obtained from advanced NMR methods. But any residual native structure in CI2 under denaturing conditions is below the limits of direct structural detection by NMR. The pKa values of the 10 acidic residues in the native state of CI2 have been measured by NMR to give indirect information.28 Most of them are 2-3 units lower than they are in model compounds. The pKa values in the acid-denatured state are, on average, 0.3 unit lower than the model compound values in pure water, but the difference disappears as ionic strength increases. This indicates some compactness in the denatured state, but it could be induced by electrostatic interactions. By all criteria, the denatured state of CI2 is a relatively expanded state. 

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

Many procedures employ 0. lit/sodium tetraborate, pH 8.5, to neutralize after the acid denaturation. This does not appear to be necessary when large volumes of PBS are used as a washing medium. 

Figure B3.6.9 Quenching of fluorescence of tumor necrosis factor (TNF) on denaturation. Fluorescence emission spectra are shown for native TNF (solid line), guanidine-unfolded TNF (dotted line), and acid-denatured TNF (dashed line). Parameters TNF concentration, 30 pg/ml Xex = 280 nm bandwidths ranging from 16 to 24 nm. Measurements made using Perkin-Elmer MPF3 spectrofluorimeter. Reprinted from Hlodan and Pain (1994) with permission of Elsevier Science.

Cook, H. W., Fatty acid denaturation and chain elongation in eucaryotes. In D. E. Vance, and J. E. Vance (eds.). Biochemistry of Lipids, Lipoproteins and Membranes. Amsterdam Elsevier Science Publishers, 1991. Provides an advanced and current treatment of fatty acid desaturation and its regulation, and cites other key references to this field. 

Figure 7.6 ESI of zinc metalloenzyme carbonic anhydrase, (a)-(c) under acidic denaturing conditions (d)-(f) native state conditions and (g) native state conditions with a specific inhibitor. Structure of entry (d) is Protein Data Bank ID IBN1. Boriack-Sjodin, P.A., Zeitlin, S., Chen, H.H., Crenshaw, L, Gross S., Dantanarayana, A., Delgado, P., May J.A., Dean, T., Christianson, D.W. Structural analysis of inhibitor binding to human carbonic anhydrase II. Protein Sciv 1998, 7, 2483-2489.

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