Structure and Dynamics of the α-Lactalbumin Molten Globule:  Fluorescence Studies Using Proteins Containing a Single Tryptophan Residue

Chakraborty, Suroopa; Ittah, Varda; Bai, Ping; Luo, Lei; Haas, Elisha; Peng, Zhengyu

doi:10.1021/bi010004w

Cited by 57 publications

(38 citation statements)

References 69 publications

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“…3). Likewise, in agreement with previous 19 F NMR and fluorescence experiments (43,44), Trp-118 is sequestered from solvent in the A and P states of HLA but accessible in the N state, indicating that the partially folded states are not simply associated with the loss of native interactions. Therefore, in addition to the nonnative tyrosine peak observed for the BLA A state, it appears that, in contrast to the situation for the main chain, there are significant nonnative environments for the side chains of aromatic residues of both BLA and HLA in their MG states.…”

Section: Discussionsupporting

confidence: 91%

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Mok

Nagashima

Day

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Photochemically induced dynamic nuclear polarization NMR pulselabeling techniques have been used to obtain detailed information about side-chain surface accessibilities in the partially folded (molten globule) states of bovine and human ␣-lactalbumin prepared under a variety of well defined conditions. Pulse labeling involves generating nuclear polarization in the partially folded state, rapidly refolding the protein within the NMR sample tube, then detecting the polarization in the well dispersed native-state spectrum. Differences in the solvent accessibility of specific side chains in the various molten globule states indicate that the hydrophobic clusters involved in stabilizing the ␣-lactalbumin fold can be formed from interactions between a variety of different hydrophobic residues in both native and nonnative environments. The multiple subsets of hydrophobic clusters are likely to result from the existence of distinct but closely related local minima on the free-energy landscape of the protein and show that the fold and topology of a given protein may be formed from degenerate groups of side chains.hydrophobic cluster ͉ protein folding ͉ pulse labeling ͉ chemically induced dynamic nuclear polarization ͉ molten globule V ery significant advances, both theoretical and experimental, have been made in recent years in elucidating the principles that govern the folding of proteins (1, 2). In particular, the concept of an energy landscape has provided a general framework for interpreting the thermodynamics and kinetics of the folding process through which a polypeptide chain converts from a disorganized unfolded state into the tightly packed native state (N state) at the global energy minimum (3, 4). Recent approaches, involving a combination of computer-simulation techniques with experimental constraints, have enabled three-dimensional structural ensembles of various partially folded species and folding intermediates to be defined and provide a coarse-grained description of the development of interresidue interactions and chain topologies involved in attaining the final native structure (5-7). Of particular importance in this context are compact denatured states, often known as molten globules (MGs), which are commonly characterized by the presence of native-like secondary structure and hydrodynamic radii but lack both native-like packing of the internal amino acid side chains and exclusion of solvent molecules from the hydrophobic core of the protein (8, 9). The detailed experimental description of such states will undoubtedly provide important information about the specific factors stabilizing the overall chain topology at a residue-specific side-chain level (10). However, MG states are very difficult to characterize by using conventional x-ray crystallography or NMR techniques because of their heterogeneous character (11,12).We have recently developed NMR techniques to characterize aromatic side chains in transient, kinetic intermediate species present in real-time protein folding experiments (13, 14) or in p...

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Section: Discussionsupporting

confidence: 91%

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Mok

Nagashima

Day

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The emission maximum for Y108W was approximately 320 nm, characteristic of a buried Trp residue, while the red-shifted emission maximum (354 nm) of Y274W is consistent with a higher degree of solvent exposure. 8,35 When a normal DNA substrate 21/30-mer was bound [DNA OH (Materials and Methods)], a significant reduction in Trp fluorescence was observed for both Y108W (30.3%) and Y274W (21.6%). Following DNA binding, dTTP incorporation into the E·DNA complex resulted in a further decrease in Trp fluorescence.…”

Section: Resultsmentioning

confidence: 99%

Conformational Dynamics of a Y-Family DNA Polymerase during Substrate Binding and Catalysis As Revealed by Interdomain Förster Resonance Energy Transfer

Maxwell

Suo

2014

Biochemistry

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Numerous kinetic, structural, and theoretical studies have established that DNA polymerases adjust their domain structures to enclose nucleotides in their active sites and then rearrange critical active site residues and substrates for catalysis, with the latter conformational change acting to kinetically limit the correct nucleotide incorporation rate. Additionally, structural studies have revealed a large conformational change between the apoprotein and the DNA–protein binary state for Y-family DNA polymerases. In previous studies [Xu, C., Maxwell, B. A., Brown, J. A., Zhang, L., and Suo, Z. (2009) PLoS Biol.7, e1000225], a real-time Förster resonance energy transfer (FRET) method was developed to monitor the global conformational transitions of DNA polymerase IV from Sulfolobus solfataricus (Dpo4), a prototype Y-family enzyme, during nucleotide binding and incorporation by measuring changes in distance between locations on the enzyme and the DNA substrate. To elucidate further details of the conformational transitions of Dpo4 during substrate binding and catalysis, in this study, the real-time FRET technique was used to monitor changes in distance between various pairs of locations in the protein itself. In addition to providing new insight into the conformational changes as revealed in previous studies, the results here show that the previously described conformational change between the apo and DNA-bound states of Dpo4 occurs in a mechanistic step distinct from initial formation or dissociation of the binary complex of Dpo4 and DNA.

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“…We observed a decrease in the labeling yield of the protein as a function of urea concentration. This does not rule out the existence of an accessibility increment but indicates that this phenomenon is coupled to the unfolding of the hydrophobic core 35,36 and that this last effect dominates the change in the labeling yield observed during this transition.…”

Section: The A-u Transition Of α-Lamentioning

confidence: 90%

Experimentally Approaching the Solvent-Accessible Surface Area of a Protein: Insights into the Acid Molten Globule of Bovine α-Lactalbumin

Craig¹,

Gómez²,

Ureta³

et al. 2009

Journal of Molecular Biology

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Structure and Dynamics of the α-Lactalbumin Molten Globule: Fluorescence Studies Using Proteins Containing a Single Tryptophan Residue

Cited by 57 publications

References 69 publications

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Conformational Dynamics of a Y-Family DNA Polymerase during Substrate Binding and Catalysis As Revealed by Interdomain Förster Resonance Energy Transfer

Experimentally Approaching the Solvent-Accessible Surface Area of a Protein: Insights into the Acid Molten Globule of Bovine α-Lactalbumin

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