Dynamic interchanging native states of lymphotactin examined by SNAPP-MS

Abstract

The human chemokine lymphotactin (Ltn) is a remarkable protein that interconverts between two unrelated native state structures in the condensed phase. It is possible to shift the equilibrium toward either conformation with selected sequence substitutions. Previous results have shown that a disulfide-stabilized variant preferentially adopts the canonical chemokine fold (Ltn10), while a single amino acid change (W55D) favors the novel Ltn40 dimeric structure. Selective noncovalent adduct protein probing (SNAPP) is a recently developed method for examining solution phase protein structure. Herein, it is demonstrated that SNAPP can easily recognize and distinguish between the Ltn10 and Ltn40 states of lymphotactin in aqueous solution. The effects of organic denaturants, acid, and disulfide bond reduction and blocking were also examined using SNAPP for the CC3, W55D, and wild type proteins. Only disulfide reduction was shown to significantly perturb the protein, and resulted in considerably decreased adduct formation consistent with loss of tertiary/secondary structure. Cold denaturation experiments demonstrated that wild-type Ltn is the most temperature sensitive of the three proteins. Examination of the higher charge states in all experiments, which are presumed to represent transition state structures between Ltn-10 and Ltn-40, reveals increased 18C6 attachment relative to the more folded structures. This observation is consistent with increased competitive intramolecular hydrogen bonding, which may guide the transition. Experiments examining the gas phase structures revealed that all three proteins can be structurally distinguished in the gas phase. In addition, the gas phase experiments enabled identification of preferred adduct binding sites.

Scheme 1

The two structures of lymphotactin

Figure 1

(a) ESI-MS spectra for wild type lymphotactin acquired in water, 50/50 water/methanol, and 49/49/1 water/methanol/acetic acid from front to back, respectively. SNAPP distributions for wild type lymphotactin in various charge states are shown in (b) +8, (c) +9, (d) +10, and (e) +12

Figure 2

SNAPP distributions for three variants of lymphotactin sampled from water. The CC3 and W55D mutants clearly produce distinguishable distributions

Scheme 2

Sequences for each lymphotactin variant

Figure 3

The average number of 18C6 adducts is shown as a function of charge state for (a) CC3, (b) wild type Ltn, and (c) W55D. The protected (disulfide reduced and capped) proteins are shown in dotted lines. (d) All proteins are shown together to demonstrate relative binding. The key is identical to (a)–(c)

Figure 4

SNAPP distributions acquired in water at room temperature and after the addition of an ice pack. Only the wild type Ltn distribution shifts to any significant extent

Figure 5

Dissociation points are shown as a function of sequence for experiments probing the gas phase structures of the +7 charge state for (a) CC3, (b) wild type Ltn, and (c) W55D (the numbering of residues for W55D has been shifted to be consistent with the other proteins, for actual numbering see Scheme 2). Differences in dissociation indicate differences in structure as described in the text