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Mechanisms of antifreeze proteins investigated via the site-directed spin labeling technique.

ABSTRACT: The site-directed spin labeling (SDSL) technique was used to examine the antifreeze mechanisms of type-I antifreeze proteins (AFPs). The effects on the growth of seed ice crystals by the spin-label groups attached to different side chains of the AFPs were observed, and the states of water molecules surrounding the spin-label groups were probed via analyses of variable-temperature (VT) dependent electron paramagnetic resonance (EPR) spectra. The first set of experiments revealed the antifreeze activities of the spin-labeled AFPs at the microscopic level, while the second set of experiments displayed those at the molecular level. The experimental results confirmed the putative ice-binding surface (IBS) of type-I AFPs. The VT EPR spectra indicate that type-I AFPs can inhibit the nucleation of seed ice crystals down to ~ - 20 °C in their aqueous solutions. Thus, the present authors believe that AFPs protect organisms from freezing damage in two ways: (1) inhibiting the nucleation of seed ice crystals, and (2) hindering the growth of seed ice crystals once they have formed. The first mechanism should play a more significant role in protecting against freezing damage among organisms living in cold environments. The VT EPR spectra also revealed that liquid-like water molecules existed around the spin-labeled non-ice-binding side chains of the AFPs frozen within the ice matrices, and ice surrounding the spin-label groups melted at subzero temperatures during the heating process. This manuscript concludes with the proposed model of antifreeze mechanisms of AFPs based on the experimental results.

SUBMITTER: Flores A

PROVIDER: S-EPMC6709975 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Mechanisms of antifreeze proteins investigated via the site-directed spin labeling technique.

Flores Antonia A Quon Justin C JC Perez Adiel F AF Ba Yong Y

European biophysics journal : EBJ 20180227 6

The site-directed spin labeling (SDSL) technique was used to examine the antifreeze mechanisms of type-I antifreeze proteins (AFPs). The effects on the growth of seed ice crystals by the spin-label groups attached to different side chains of the AFPs were observed, and the states of water molecules surrounding the spin-label groups were probed via analyses of variable-temperature (VT) dependent electron paramagnetic resonance (EPR) spectra. The first set of experiments revealed the antifreeze ac ...[more]

PMID: 29487966

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Project description:Protein structures are not static but sample different conformations over a range of amplitudes and time scales. These fluctuations may involve relatively small changes in bond angles or quite large rearrangements in secondary structure and tertiary fold. The equilibrium between discrete structural substates on the microsecond to millisecond time scale is sometimes termed conformational exchange. Protein dynamics and conformational exchange are believed to provide the basis for many important activities, such as protein-protein and protein-ligand interactions, enzymatic activity and protein allostery; however, for many proteins, the dynamics and conformational exchange that lead to function are poorly defined. Spectroscopic methods, such as NMR, are among the most important methods to explore protein dynamics and conformational exchange; however, they are difficult to implement in some systems and with some types of exchange events. Site-directed spin labeling (SDSL) is an EPR based approach that is particularly well-suited to high molecular-weight systems such as membrane proteins. Because of the relatively fast time scale for EPR spectroscopy, it is an excellent method to examine exchange. Conformations that are in exchange are captured as distinct populations in the EPR spectrum, and this feature when combined with the use of methods that can shift the free energy of conformational substates allows one to identify regions of proteins that are in dynamic exchange. In addition, modern pulse EPR methods have the ability to examine conformational heterogeneity, resolve discrete protein states, and identify the substates in exchange. Protein crystallography has provided high-resolution models for a number of membrane proteins; but because of conformational exchange, these models do not always reflect the structures that are present when the protein is in a native bilayer environment. In the case of the Escherichia coli vitamin B12 transporter, BtuB, the energy coupling segment of this protein undergoes a substrate-dependent unfolding, which acts to couple this outer-membrane protein to the inner-membrane protein TonB. EPR spectroscopy demonstrates that the energy coupling segment is in equilibrium between ordered and disordered states, and that substrate binding shifts this equilibrium to favor an unfolded state. However, in crystal structures of BtuB, this segment is resolved and folded within the protein, and neither the presence of this equilibrium nor the substrate-induced change is revealed. This is a result of the solute environment and the crystal lattice, both of which act to stabilize one conformational substate of the transporter. Using SDSL, it can be shown that conformational exchange is present in other regions of BtuB and in other members of this transporter family. Conformational exchange has also been examined in systems such as the plasma membrane SNARE protein, syntaxin 1A, where dynamics are controlled by regulatory proteins such as munc18. Regulating the microsecond to millisecond time scale dynamics in the neuronal SNAREs is likely to be a key feature that regulates assembly of the SNAREs and neurotransmitter release.

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Mechanisms of antifreeze proteins investigated via the site-directed spin labeling technique.

Publications

Mechanisms of antifreeze proteins investigated via the site-directed spin labeling technique.

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