Proteins in Solution

A protein may adopt in solution various structures with more or less subtle differences, depending on temperature, protein concentration, pH, ionic strength or addition of co-solvent or lipids. The rapid FTIR analysis is well suited to probe the extent in secondary structures of the protein in the solution state used as reference for the adsorption experiments.

As an illustration, the lists of the amide I' component bands identified via second-derivative methods are reported in Table 3 for BSA and lysozyme in deuterated phosphate buffer (Noinville et al. 2002; Servagent-Noinville et al. 2000). For a-helical proteins such as BSA, our infrared spectroscopic analysis distinguishes a major group of regular helices giving a component band at 1,651 cm-1 from a minor group of irregular helices at 1,660 cm-1. This latter component band at a higher wavenumber is attributed to helices packed in a more hydrophobic environment (Dousseau and Pezolet 1990; Rothshild and Clark 1979). Our results show that BSA is stabilised by a large amount of a-helical domains, representing 53% of the overall polypeptide backbone at pD 7.5 (Table 3). The x-ray structure of homologous serum albumins such as human serum albumin (HAS) gives approximately 67% of the a-helix (Carter and Ho 1994). The stability of the secondary structure of BSA is known to depend on pH. Previous infrared analysis based

Table 3. Assignments of the amide I' components and extent in secondary structure elements (expressed as a percentage of overall amide I' area) of BSA and lysozyme incubated for 4 h in deuterated phosphate buffer at pD 7.5

Assignments to BSA Lysozyme

Table 3. Assignments of the amide I' components and extent in secondary structure elements (expressed as a percentage of overall amide I' area) of BSA and lysozyme incubated for 4 h in deuterated phosphate buffer at pD 7.5

Assignments to BSA Lysozyme

v(CO) peptide carbonyls

Wave number (cm-1)

Area (%)

Wave number (cm-1)

Area (%)

Random domains

1681

1689

12

18

Random domains

1671

1670

Hydrophobic a-helices

1660

14

-

-

a-helices

1651

39

1652

34

Hydrated random domains

1640

13

1641

27

Bent, ^-strands or ^-sheets

1630

23

1630

11

Self-association

1612

2

1610

10

Reference

(Servagent-Noinville

(Noinville et al. 2002)

et al. 2000)

on the same spectral decomposition predicted a61% helix content for BSA at a pD equal to its isoelectric point (Servagent-Noinville et al. 2000). The intramolecular hydrogen bonds, visible at 1,630 cm-1 involving approximately 23% of the peptide groups correspond to bent domains in the BSA backbone. This is in agreement with secondary structure predictions estimating that around 23% of the structure in extended chain conformation would be predicted as ^-strand (Carter and Ho 1994). In the random coil domains, BSA presents 13% of its carbonyl groups from peptide bonds involved in hydrogen bonding with water (1,640 cm-1). Other random coil domains represent 12% of the BSA backbone, and self-associated domains only 2% (Table 3). The component band located in the 1,610-1,620 cm-1 spectral region appears mainly after denaturation of a protein, as reported for cytochrome c (Muga et al. 1991) and for lectins (Wantyghem et al. 1990). This low wavenumber component is attributed to peptide CO, which is involved in intermolecular ^-sheets or in self-association or aggregation, and is often found in amyloid peptides or in oligomeric prions (Revault et al. 2005; Sokolowski et al. 2003).

While the crystal structure of lysozyme reveals that 28% of the residues are in a-helical conformation (Wilson et al. 1992), the FTIR analysis gives an a-helix content of 34%. The crystallised lysozyme also contains a relatively high amount of 3i0-helix (11%). If we assume that the absorption frequencies of 3io-helix and a-helix are closed enough to form only one component band at around 1,650 cm-1, the helical content in lysozyme reaches 39%. Different infrared absorption frequencies for this infrequent 310-helical conformation in globular proteins have been found in the literature. It has been proposed that 310-helices absorb at a lower frequency than a-helices in D2O solutions due to their tighter geometry (Prestrelski et al. 1991), rather than at 1,665 cm-1 as previously reported (Krimm and Bandekar 1986). The amount of hydrogen-bonded peptide CO with water molecules found for lysozyme is rather large (around 27%). Following the attribution given by Prestrelski et al., some of this H-bonded peptide CO absorbing at 1,640 cm-1 would in factbe involved ina310-helix. Concerning ^-structure, only 6% of the residues in the crystalline state of lysozyme are in ^-strands (Wilson et al. 1992), whereas 11% of H-bonded peptide groups involved in ^-structure are found by FTIR. Some of the peptide carbonyls considered as turns (35%) or as bends (4.6%) in the secondary structure from the x-ray data are interleaved between peptide CO involved in ^-strands so that they could either contribute to the infrared band at 1,630 cm-1 if hydrogen-bonded, or to the infrared band attributed to peptide CO involved in loops in polar environments at 1,670 cm-1 if non-hydrogen bonded (Wantyghem etal. 1990).

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