Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide

Abstract

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in beta B1-crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble beta B1 contained 13%-17% deamidation at Gln146 and Asn157. Static and quasi-elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild-type (wt) beta B1 and the deamidated beta B1 mutants, Q146E and N157D. Dimer formation occurred for wt beta B1, Q146E, and N157D in a concentration-dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of beta B1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.

Figure 1.

Tandem mass spectra of (A) esterified tryptic peptide 143–149 of βB1-crystallin containing a deamidated Gln146 and (B) esterified tryptic peptide 150–159 of βB1-crystallin containing a deamidated Asn157 from the soluble protein of a 55-year-old human lens. The parent ion of peptide 143–149 was singly charged with m/z = 927.6 (expected = 927.5), and the parent ion of peptide 150–159 was doubly charged with m/z = 589.6 (expected 589.4). The observed y and b fragment ions are indicated in the spectra. The asterisk indicates the position of the deamidated residue.

Figure 2.

Extracted ion chromatograms for both the singly-charged amidated (m/z range 909.0–910.0, peak at 18.8 min) and singly charged deamidated (m/z range 927.1–928.1, peak at 25.8 min) forms of peptide 143–149 of βB1-crystallin from soluble proteins of 55-year-old lenses. The 19.1% deamidation of peptide 143–149 was calculated by dividing the area under the peak for the deamidated peptide (626) by the total areas of both peaks (3276) × 100.

Figure 3.

(A) Far-UV and (B) near-UV circular dichroism of wt βB1 (blue spectrum), Q146E (green spectrum), and N157D (red spectrum). Protein concentrations ranged from 1.1 to 1.6 mg/mL. All spectra were averaged over 30 scans.

Figure 4.

Chromatograms of (A) the molar masses and (B) hydrodynamic radii of whole soluble protein extracted from lenses from a 3-month-old donor. The line tracing represents the signal from the refractive index detector. In (A), the filled diamonds represent the molar masses. In (B), the filled diamonds represent the hydrodynamic radii. A 50-μL sample of 15 mg/mL whole soluble protein was analyzed.

Figure 5.

Chromatograms of the molar masses for the recombinant proteins (A) wt βB1, (B) N157D, and (C) Q146E. Filled diamonds represent the molar masses determined for each eluting slice. The line represents the refractive index signal; all refractive index tracings are normalized to one another. The concentration of each protein was 10 mg/mL in a 50-μL sample.

Figure 6.

Thermal denaturation/precipitation curves. Thermal denaturation curves of wt βB1 (middle line), N157D (bottom line), and Q146E (top line) were obtained by heating 0.1 mg/mL concentration of proteins at 55°C and measuring change in turbidity at 405 nm. Data shown for wt were the average of two experiments, and are similar to previously reported results (Lampi et al. 2002). Data shown for N157D and Q146E were the average of four to five experiments. Samples were also heated at 1.5 mg/mL and visualized by SDS-PAGE (inset: S, supernatant; P, pellet; MW, molecular weight marker).

Figure 7.

Diagram of a Kirkwood approximation of a truncated βB1 dimer using an offset arrangement of spheres (A–D). Each sphere represents a domain. The distance between A and B, C and D, and A and D is 2R_s, where R_s is the radius of the sphere. The distance between B and C is (square root of 5)R_s.