Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is employed here to describe equilibrium protein conformational transitions and to analyze the influence of instrumental settings, pH, and solvent surface tension on the charge-state distributions (CSD). A first set of experiments shows that high flow rates of N(2) as curtain gas can induce unfolding of cytochrome c (cyt c) and myoglobin (Mb), under conditions in which the stability of the native protein structure has already been reduced by acidification. However, it is possible to identify conditions under which the instrumental settings are not limiting factors for the conformational stability of the protein inside ESI droplets. Under such conditions, equilibrium unfolding transitions described by ESI-MS are comparable with those obtained by other established biophysical methods. Experiments with the very stable proteins ubiquitin (Ubq) and lysozyme (Lyz) enable testing of the influence of extreme pH changes on the ESI process, uncoupled from acid-induced unfolding. When HCl is used for acidification, Ubq and Lyz mass spectra do not change between pH~7 and pH 2.2, indicating that the CSD is highly characteristic of a given protein conformation and not directly affected by even large pH changes. Use of formic or acetic acid for acidification of Ubq solutions results in major spectral changes that can be interpreted in terms of protein unfolding as a result of the increased hydrophobicity of the solvent. On the other hand, Lyz, cyt c, and Mb enable direct comparison of protein CSD (corresponding to either the folded or the unfolded protein) in HCl or acetic acid solutions at low pH. The values of surface tension for these solutions differ significantly. Confirming indications already present in the literature, we observe very similar CSD under these solvent conditions for several proteins in either compact or disordered conformations. The same is true for comparison between water and water-acetic acid for folded cyt c and Lyz. Thus, protein CSD from water-acetic solutions do not seem to be limited by the low surface tension of acetic acid as previously suggested. This result could reflect a general lack of dependence of protein CSD on the surface tension of the solvent. However, it is also possible that the effect of acetic acid on the precursor ESI droplets is smaller than generally assumed.
S134N copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis. Earlier studies demonstrated that partially metaldeficient S134N SOD1 crystallized in filament-like arrays with abnormal contacts between the individual protein molecules. Because protein aggregation is implicated in SOD1-linked familial amyotrophic lateral sclerosis, abnormal intermolecular interactions between mutant SOD1 proteins could be relevant to the mechanism of pathogenesis in the disease. We have therefore applied NMR methods to ascertain whether abnormal contacts also form between S134N SOD1 molecules in solution and whether Cys-6 or Cys-111 plays any role in the aggregation. Our studies demonstrate that the behavior of fully metallated S134N SOD1 is dramatically different from that of fully metallated wild type SOD1 with a region of subnanosecond mobility located close to the site of the mutation. Such a high degree of mobility is usually seen only in the apo form of wild type SOD1, because binding of zinc to the zinc site normally immobilizes that region. In addition, concentration-dependent chemical shift differences were observed for S134N SOD1 that were not observed for wild type SOD1, indicative of abnormal intermolecular contacts in solution. We have here also established that the two free cysteines (6 and 111) do not play a role in this behavior.Over 100 different mutations in the superoxide dismutase 1(SOD1) 4 gene have been linked to the inherited form of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by progressive death of motor neurons and consequent paralysis. The individual mutations have been shown to exert their pathological effects by a gain of function mechanism, implying that the copper-zinc superoxide dismutase variant (Cu,Zn-SOD) expressed from the mutated gene has in some way become toxic. Recent evidence suggests that SOD1-linked ALS, like many other neurodegenerative diseases, is a protein misfolding disorder characterized by abnormal deposits of aggregated proteins in neural tissues (1-3). Implicated also in the toxicity of ALS-mutant SOD1 proteins are accelerated oxidative damage and inhibition of proteasome, chaperone, or mitochondrial function (2, 4, 5).Relatively large, insoluble proteinaceous inclusions are observed in neural tissue in ALS and other neurodegenerative diseases, but it has been proposed that, rather than causing the disease, these deposits of aggregated protein may be formed as a part of a defensive mechanism to sequester misfolded and oligomerized protein when excessive amounts accumulate. More recently it has been suggested that the toxic species are smaller, high molecular weight oligomerized proteins formed from abnormal contacts of misfolded proteins resulting in protofibrils, pores, or other toxic species (6 -10). High molecular weight forms of oligomerized mutant SOD1 have been observed in several studies using ALS-SOD1 transgenic mice (11-13).Many of the ALS-linked Cu,Zn-S...
The enzyme NCS [(S)-norcoclaurine synthase; EC 4.2.1.78] found in the common meadow rue, Thalictrum flavum, and other plant species, is involved in the biosynthesis of BIAs (benzylisoquinoline alkaloids). This group of plant secondary metabolites comprises pharmacologically-active compounds such as morphine and codeine. NCS catalyses the condensation of 4-HPAA (4-hydroxyphenylacetaldehyde) and dopamine to (S)-norcoclaurine, the common precursor of all plant BIAs. Although enzymatic properties of NCS and mechanistic aspects of the reaction have been studied in detail, no structural information on NCS was available so far. The enzyme shows significant sequence homology to members of the PR10 proteins (class 10 of pathogenesis-related proteins) such as the major birch pollen allergen Bet v 1. Our CD and NMR spectroscopic data indicated high similarity of the NCS and the Bet v 1 fold and allowed us to model NCS using Bet v 1 as a template. Virtually complete backbone assignment of the NCS sequence was used to study substrate binding by NMR titration experiments. Although binding of 4-HPAA seems to induce side-chain rearrangements in an extensive part of the protein, the putative distinct interaction site for dopamine could be clearly identified. The oligomerization state of NCS that reportedly plays an important role in enzyme functionality was determined to be concentration-dependent by SEC (size-exclusion chromatography) as well as NMR relaxation measurements, and the enzyme was found to be predominantly a monomer at the low micromolar concentrations used for activity assays.
Electrospray ionization mass spectrometry (ESI-MS) applied to protein conformational studies is a powerful new method that seems to provide specific information about protein tertiary structure. In this study, we analyzed the effect of trifluoroethanol (TFE) on a myoglobin peptide and cytochrome c (cyt c) at low pH by circular dichroism (CD) and ESI-MS. These experiments show that coil-to-helix transition per se does not affect ESI mass spectra, confirming that this technique is insensitive to the local conformation of the polypeptidic chain and, rather, reports on the tertiary contacts characterizing different protein conformations. This property makes ESI-MS an excellent method, complementary to CD, for the characterization of protein conformational changes. Fluorinated alcohols have been suggested to induce molten globule formation in acid-unfolded cyt c. The experiments described here show that TFE does not induce major changes in the ESI mass spectrum of cyt c at pH 2.2, indicating that no stabilization of compact, globular structures is detectable under the conditions employed. On the other hand, even low concentrations of TFE (2-5%) are shown to destabilize the folded state of the protein around the mid-point of its acid-induced unfolding transition.
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