Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Feb;13(2):332-41.
doi: 10.1110/ps.03180004.

Thermal stability of human alpha-crystallins sensed by amide hydrogen exchange

Azeem Hasan  1 , Jiong Yu, David L Smith, Jean B Smith
Affiliations

Thermal stability of human alpha-crystallins sensed by amide hydrogen exchange

Azeem Hasan et al. Protein Sci. 2004 Feb.

Abstract

The alpha-crystallins, alphaA and alphaB, are major lens structural proteins with chaperone-like activity and sequence homology to small heat-shock proteins. As yet, their crystal structures have not been determined because of the large size and heterogeneity of the assemblies they form in solution. Because alpha-crystallin chaperone activity increases with temperature, understanding structural changes of alpha-crystallin as it is heated may help elucidate the mechanism of chaperone activity. Although a variety of techniques have been used to probe changes in heat-stressed alpha-crystallin, the results have not yet yielded a clear understanding of chaperone activity. We report examination of native assemblies of human lens alpha-crystallin using hydrogen/deuterium exchange in conjunction with enzymatic digestion and analysis by mass spectrometry. This technique has the advantage of sensing structural changes along much of the protein backbone and being able to detect changes specific to alphaA and alphaB in the native assembly. The reactivity of the amide linkages to hydrogen/deuterium exchange was determined for 92% of the sequence of alphaA and 99% of alphaB. The behavior of alphaA and alphaB is remarkably similar. At low temperatures, there are regions at the beginning of the alpha-crystallin domains in both alphaA and alphaB that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of alphaA also has low protection. With increasing temperatures, both proteins show gradual unfolding. The maximum percent change in exposure with increasing temperatures was found in alphaA 72-75 and alphaB 76-79, two regions considered critical for chaperone activity.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
HPLC ESI mass spectra of α-crystallins labeled with deuterium under different conditions. (A) No exposure to D2O. (B) Exposed to D2O at 37°C for 112 sec. (C) Completely exchanged in 95% D2O. αA-S and αB + P refer to αA that has lost the C-terminal Ser and phosphorylated αB, respectively.
Figure 2.
Figure 2.
Deuterium levels found in four α-crystallins following exposure to D2O for various times (t1–5) at 37°C, 42°C, 47°C, 52°C, and 57°C. See Table 1 for values of t1–5.
Figure 3.
Figure 3.
Amino acid sequences and peptic fragments of human αA- and αB-crystallin. All peptides were identified by their molecular masses and CID MS/MS. Numbers imbedded in the arrows give the monoisotopic molecular mass of each peptide.
Figure 4.
Figure 4.
Mass spectra of peptides including residues 28–37 (singly charged) and 144–175 (quadruply charged) of αB-crystallin. (A) No exposure to D2O. (B,C) Exposed to D2O at 27°C for 71 sec and 1136 sec, respectively. (D) Completely exchanged in 95% D2O.
Figure 5.
Figure 5.
Deuterium levels found in peptides of αA-crystallin (A) and αB-crystallin (B) following exposure of the native protein to D2O for various times at 27°C. The deuterium levels were normalized to the deuterium levels found in the fragment derived from α-crystallin equilibrated in 95% D2O.
Figure 6.
Figure 6.
Deuterium levels found in peptic fragments of α-crystallin following exposure of the intact protein to D2O at different temperatures for different times. (A) αA-Crystallin, short (t1) exposure time. (B) αA-Crystallin, long (t5) exposure time. (C) αB-Crystallin, short (t1) exposure time. (D) αB-Crystallin, long (t5) exposure time. The exposure time used for each temperature was adjusted to compensate for the temperature dependence of the rate of intrinsic hydrogen exchange (see Table 1).
Figure 7.
Figure 7.
Deuterium levels in peptic fragments of α-crystallin when the protein was exposed to D2O at 37°C for 28 sec. Prior to labeling, the protein was equilibrated at 37°C for 30 min (open bars) or heated to 67°C for 30 min, then cooled to 37°C (closed bars).
Figure 8.
Figure 8.
The difference between deuterium levels found in peptic fragments of αA- and αB-crystallins following exposure of the intact protein to D2O for 57°C or 27°C, both at t1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Abgar, S., Backman, J., Aerts, T., Vanhoudt, J., and Clauwaert, J. 2000. The structural differences between bovine lens αA- and αB-crystallin. Eur. J. Biochem. 267 5916–5925. - PubMed
    1. Bai, Y.W., Milne, J.S., Mayne, L., and Englander, S.W. 1993. Primary structure effects on peptide group hydrogen exchange. Proteins 17 75–86. - PMC - PubMed
    1. ———. 1994. Protein stability parameters measured by hydrogen exchange. Proteins 20 4–14. - PubMed
    1. Berengian, A.R., Bova, M.P., and Mchaourab, H.S. 1997. Structure and function of the conserved domain in αA-crystallin. Site-directed spin labeling identifies a β-strand located near a subunit interface. Biochemistry 36 9951–9957. - PubMed
    1. Berghuis, A.M. and Brayer, G.D. 1992. Oxidation state-dependent conformational changes in cytochrome c. J. Mol. Biol. 223 959–976. - PubMed

Publication types