The transition from the native to the acid-state characterized by multi-spectroscopy approach: Study for the holo-form of bovine α-lactalbumin

Adriana Litwińczuk, Soo Ryeon Ryu, Laurence A. Nafie, Jong Wha Lee, Hugh I. Kim, Young Mee Jung, Bogusława Czarnik-Matusewicz

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22 Citations (Scopus)


The transition of the holo-form of bovine α-lactalbumin from the native (N) to the pH-generated acidic-state (A-state) was analyzed by probing its tertiary and secondary structure using a concerted spectroscopic approach combining near- and far-UV circular dichroism (CD), electrospray ionization ion mobility mass spectrometry (ESI-IM-MS), vibrational circular dichroism (VCD), and Fourier transform infrared spectroscopy (FTIR) in the attenuated total reflection (ATR) and transmission (TR) modes. The spectroscopic results, which relied on the interaction of an electromagnetic field with different molecular targets, confirmed the decay of extensive rigid side-chain packing interactions during the pH-induced N → A-state transition and revealed the targets' dependence on secondary structural changes. Independent analyses of the spectral changes using two methods of multivariate analysis, such as principal component analysis and two-dimensional correlation spectroscopy, revealed small but significant differences in the secondary structure as a result of the all-or-none transition. The cooperativity of the transition was quantitatively described using values corresponding to the mid-point (tm) and width of the transition (Δtm). The averages of the two parameters, calculated using the data collected by the different probes, were equal to 3.5 ± 0.2 and 0.6 ± 0.1(SE), respectively. The variable two-state nature of the cooperative N → A-state transition confirmed that the protonation of the side chain carboxyl groups on the Asp and Glu residues and that the release of a Ca2 + ion induced structural changes on both the secondary and tertiary levels. The changes have been confirmed by results obtained from the concerted spectroscopic approach.

Original languageEnglish
Pages (from-to)593-606
Number of pages14
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Issue number3
Publication statusPublished - 2014 Mar
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the National Research Foundation of Korea (NRF) grants (No. NRF-2012R1A1A3011844 and 2009–0087013 ) and the Korean Federation of Science and Technology Societies Grant funded by the Korean government (MEST) . BCM is grateful to the National Science Centre for research funding (grant no. 2012/05/B/ST/02029 ) and AL to the University of Wrocław for grant no 2267/M/WCH/12 .


  • A-state molten globule
  • Holo α-lactalbumin
  • Multi-spectral analysis
  • Principal component analysis
  • Protein structure
  • Two-dimensional correlation spectroscopy

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology


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