Probing Protein Structure by Solvent Perturbation of NMR Spectra

Improta, Sabina; Molinari, Henríette; Pastore, Annalisa; Consonni, Roberto; Zetta, Lucia

doi:10.1111/j.1432-1033.1995.tb20362.x

Cited by 25 publications

(17 citation statements)

References 27 publications

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“…In the present paper, structural characterization of the aromatic domain of the recombinant P2 has been performed by NMR and photo-C1DNP spectroscopies. In recent years the photo-CIDNP technique has been used to investigate a large number of proteins [10][11][12][13]. The photo-CIDNP effect arises from the nuclear spin polarization generated in a reversible photochemical reaction ofa flavin dye with tyrosines, tryptophans and histidines on the protein surface.…”

Section: Introductionmentioning

confidence: 99%

¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

Consonni¹,

Limiroli

Molinari

et al. 1995

FEBS Letters

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Section: Introductionmentioning

confidence: 99%

¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

Consonni¹,

Limiroli

Molinari

et al. 1995

FEBS Letters

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“…Trp60 appears partially exposed to TEMPOL, but as mentioned above, its exposure could not be judged from photo-CIDNP spectra because of overlap. Tyrl03 is exposed to TEMPOL but not to the dye at 300 K although it appears in the photo-CIDNP spectrum after increasing the temperature (above 320 K, Improta et al, 1994). To explain this result, an inspection of the model obtained from the X-ray structure of baboon a-lactalbumin shows that the hydroxyl group of Tyrl03 is involved in hydrogen bonding with the backbone oxygen of Gln54, even though the 6 and E protons are fully exposed.…”

Section: Discussionmentioning

confidence: 99%

“…Dipolar interactions between the electron spin on the probe and the nearby nuclear spins in the protein cause line broadening of the NMR spectrum, which could lead to the loss of the cross-peak if the intrinsic linewidth is already broad. This can be of specific relevance in exchange studies (Improta et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

“…This combined approach may therefore be valuable in applications where it is otherwise difficult to map protein surfaces as in the study of protein folding intermediates (Improta et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we report the control experiments on the native state of bovine a-lactalbumin. In the following study, we present results obtained by combining information from photo-CIDNP and spin label perturbation to the study of protein folding (Improta et al, 1994).…”

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Probing Protein Structure by Solvent Perturbation of NMR Spectra

Improta

Molinari

Pastore

et al. 1995

European Journal of Biochemistry

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We have suggested elsewhere the use of surface mapping by spin label probes (Esposito et al., 1992). According to this approach, soluble nitroxides are added to a protein solution. Resonances of protons that are accessible to the nitroxide are broadened and bleached out of the spectrum, while resonances in the protein interior remain unaffected. This approach is, in principle, complementary to another technique, photochemically induced dynamic nuclear polarization, which maps the position of aromatic protons on the protein surface. A detailed comparison between the two techniques is necessary for a confident use of the more recent suggested nitroxide perturbation approach. In the present study, we show that the results obtained by the two techniques for the native state of bovine a-lactalbumin are fully consistent and may therefore be combined for the study of protein surfaces.Keywords. Surface mapping ; NMR ; photo-CIDNP ; TEMPOL.The knowledge of which residues in a protein are exposed to the solvent or accessible to it, and which other residues are buried is important for structure determination, studies of intermolecular interactions and protein dynamics. Several spectroscopic techniques have been applied to map the protein surface without prior knowledge of the complete three-dimensional structure. Fluorescence spectroscopy is for instance a widely applied technique very sensitive to the environment of aromatic side chains (Creighton, 1993). Both the wavelength of emitted light and the quantum yield of fluorescence spectra may differentiate between an aromatic residue buried in the protein interior or exposed on the surface. However, limitations may arise from the number and the nature of the aromatic groups present. If both tryptophan and tyrosine residues are present, for instance, the quantum yield of the former may mask completely the contribution of the latter. Furthermore, the intrinsic resolution of the fluorescence spectrum does not allow discrimination among aromatic residues of the same type. Although very useful qualitatively, in a general case this method fails to give detailed information on specific sites.This local information might be provided instead by photochemically induced dynamic nuclear polarization (photo-CIDNP) experiments if at least partial assignment of an NMR spectrum is available (Kaptein et al

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1,1,1,3,3,3‐hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine α‐lactalbumin: Calorimetric and spectroscopic studies

Kundu

Kishore

2004

Biopolymers

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The thermal denaturation of alpha-lactalbumin was studied at pH 7.0 and 9.0 in aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) by high-sensitivity differential scanning calorimetry. The conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. The most obvious effect of HFIP was lowering of the transition temperature with an increase in the concentration of the alcohol up to 0.30M, beyond which no calorimetric transition was observed. Up to 0.30M HFIP the calorimetric and van't Hoff enthalpy remained the same, indicating the validity of the two-state approximation for the thermal unfolding of alpha-lactalbumin. The quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. Spectroscopic observations confirm that alpha-lactalbumin is in the molten globule state in the presence of 0.50M HFIP at pH 7.0 and 0.75M HFIP at pH 9.0. The results also demonstrate that alpha-lactalbumin in the molten globule state undergoes a noncooperative thermal transition to the denatured state. It is observed that two of four tryptophans are exposed to the solvent in the HFIP induced molten globule state of alpha-lactalbumin compared to four in the 8.5M urea induced denatured state of the protein. It is also observed that the HFIP induced molten globule states at the two pH values are different from the acid induced molten globule state (A state) of alpha-lactalbumin.

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Probing Protein Structure by Solvent Perturbation of NMR Spectra

Cited by 25 publications

References 27 publications

¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

Probing Protein Structure by Solvent Perturbation of NMR Spectra

1,1,1,3,3,3‐hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine α‐lactalbumin: Calorimetric and spectroscopic studies

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Probing Protein Structure by Solvent Perturbation of NMR Spectra

Cited by 25 publications

References 27 publications

1H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp23 and Phe31 in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

1H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp23 and Phe31 in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

Probing Protein Structure by Solvent Perturbation of NMR Spectra

1,1,1,3,3,3‐hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine α‐lactalbumin: Calorimetric and spectroscopic studies

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¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus