Protein sequences bound to mineral surfaces persist into deep time

Abstract

Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C).

Keywords: Struthio camelus; biochemistry; biomineralization; eggshell; evolutionary biology; genomics; molecular dynamics; paleontology; paleoproteomics.

Figure 1.. Eggshell peptide sequences from Africa have thermal ages two orders of magnitude older than those reported for DNA or bone collagen.

(A) Sites reporting the oldest DNA and collagen sequences are from high latitude sites compared to ostrich eggshell samples from sites in Africa illustrated in (B) for which the current mean annual air temperatures are much higher. (C) Kinetic estimates of rates of decay for DNA (Lindahl and Nyberg, 1972), collagen (Buckley and Collins, 2011) and ostrich eggshell proteins (Crisp et al., 2013) were used to estimate thermal age assuming a constant 10°C (Figure 1—source data 1; Appendix 1. For Elands Bay Cave and Pinnacle Point the oldest samples are shown). Note log scale on the z-axis: struthiocalcin-1 peptide survival is two orders of magnitude greater than any previously reported sequence, offering scope for the survival of peptide sequences into deep time. DOI: http://dx.doi.org/10.7554/eLife.17092.003

Figure 2.. Proteome persistence and patterns of degradation.

(A) Amino acid racemization in ostrich eggshell up to 3.8 Ma old from sites in South Africa and Tanzania. (B) Linear increase of THAA Val D/L values with the log of thermal age. (C) Exponential decrease of the number of identified MS/MS spectra with age (THAA Val D/L). (D) The average hydropathicity of the peptides identified remains stable up to Val D/Ls ~1. Note that Val D/Ls > 1.0 are unexpected and may be due to decomposition processes occurring in the closed system. The intracrystalline proteome composition in modern eggshell does not vary across microstructural layers (Figure 2—figure supplement 1), but patterns of degradation are different between fossil samples and purified proteins degraded at high temperature in the absence of the mineral (Figure 2—figure supplement 2). DOI: http://dx.doi.org/10.7554/eLife.17092.005

Figure 2—figure supplement 1.. Structure and composition of OES.

(A) modern (left) and fossil (LOT 13898; right) OES: crystalline (1), prismatic (2), cone (3) and organic (4) layers. (B) comparison of total THAA yields in each layer before and after bleaching. (C) comparison between the composition of bleached eggshell powder from the cone, palisade and crystalline layers. DOI: http://dx.doi.org/10.7554/eLife.17092.006

Figure 2—figure supplement 2.. Proteome degradation.

(A–B) fossil OES: (A) number of unique proteins; (B) mean peptide length (excluding contaminants). (C–E) degradation of purified proteins in water: (C) number of unique proteins identified; (D) number of identified product ion spectra; (E) mean peptide length; (F) average hydropathicity. No peptides were detected in the 120 hr heated sample. DOI: http://dx.doi.org/10.7554/eLife.17092.007

Figure 3.. Survival of struthiocalcin 1 and struthiocalcin 2 peptides.

Over time (and increasing THAA Val D/L values) the spectral count decreases as degradation progresses. Blue bars highlight the peptides investigated computationally (represented by the filled atoms in the models). Highly degraded samples (Val D/L ~0.8–1.1) preserve the DDDD-containing peptide. The full time series is shown for SCA-1 in Figure 3—figure supplement 1 and for SCA-2 in Figure 3—figure supplement 2. DOI: http://dx.doi.org/10.7554/eLife.17092.015

Figure 3—figure supplement 1.. Frequency of identified spectra of SCA-1 in bleached OES (fossils) and purified proteins (kinetics).

Spectral count scale: 0–400 for fossil OES; 0–200 for kinetics. Sample 4605 has been recognized as burnt (Crisp, 2013) but excellent sequence preservation is observed. Low spectral counts for sample 4613 are likely due to sample preparation, as AAR did not identify this sample as problematic. Coloured bars show protein structure. DOI: http://dx.doi.org/10.7554/eLife.17092.016

Figure 3—figure supplement 2.. Frequency of identified spectra of SCA-2 in bleached OES (fossils) and purified proteins (kinetics).

Spectral count scale: 0–400 for fossil OES; 0–200 for kinetics. Sample 4605 has been recognized as burnt (Crisp 2013) but excellent sequence preservation is observed. No SCA-2 peptides were found for sample 4613; this is likely due to sample preparation. Wonderwerk and Laetoli samples yielded some peptide sequence, but not consistently. DOI: http://dx.doi.org/10.7554/eLife.17092.017

Figure 4.. Authenticity of the ancient sequences.

Amino acid analyses (A): Total concentrations in all eggshell samples (sum of Asx, Glx, Gly, Ala, Val and Ile). Carry-over: (B) Total ion chromatogram for one eggshell sample (EBC_1823) and the blank analysed immediately after (blank_post_EBC1823). (C) Spectral abundance of SCA-1 and SCA-2 in LC-MS/MS blanks. (D) SCA-1 coverage in the blank analysed after a Pinnacle Point eggshell sample PP_4652. Note 'DDDD-' and 'EEEED-' peptides and Asn deamidation. (E) Extracted ion chromatogram for LDDDDYPK in EBC_1823, blank_post_EBC1823 and EBC_1819. (F) Absolute and relative total abundance of 'DDDD' peptides in Laetoli samples/blanks. Signal reduction is at least 100-fold (more often 1000- or 10,000-fold). Independent replication and manual de novo sequencing of the peptides from Laetoli (Appendix 5, section A; Supplementary file 2), consistency of diagenesis-induced modifications (Appendix 5, section D; Supplementary file 3) and volatile organic compound analyses (Appendix 5, section E) were also used to validate the results obtained. DOI: http://dx.doi.org/10.7554/eLife.17092.018

Figure 5.. Schematic diagram of energy barriers for peptide hydrolysis.

A pictorial representation of the energy barriers associated with the lysis of the peptide. The process in bulk water is depicted in red and the process at the surface is depicted in blue. The surface process shows a larger barrier due to the stabilization of the reactants at the surface. DOI: http://dx.doi.org/10.7554/eLife.17092.019