Dynamic Interchanging Native States of Lymphotacti

Authors: Qingyu Sun Robert C Tyler Brian F Volkman Ryan R Julian

Publish Date: 2011/01/15

Volume: 22, Issue: 3, Pages: 399-407

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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 disulfidestabilized 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 wildtype 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 Ltn10 and Ltn40 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 sitesSelective noncovalent adduct protein probing SNAPP is a recently developed method for examining protein structure in solution 4 This method relies on specific noncovalent interactions to probe protein structure Experiments are conducted by introducing a reagent which binds weakly in solution such as 18crown6 ether 18C6 which becomes strongly attached to the protein in the gas phase 18C6 attaches to lysine in the gas phase due to three specific hydrogen bonds between the protonated sidechain amine and alternating oxygen atoms in the crown ether The binding energy for this attachment is ~54 kcal/mol 5 Less specific although fairly strong noncovalent 42 kcal/mol associations between 18C6 and the protonated side chain of arginine can form as well SNAPP experiments are conducted by subjecting an aqueous solution containing protein and 18C6 to electrospray ionization ESI Source conditions which favor both complete desolvation and retention of noncovalent adducts must be employed During the process of ESI binding interactions between 18C6 and protonated side chains transition from weak to strong as the protein is rapidly desolvated Previous experiments have determined that the local chemical environment surrounding each lysine residue strongly influences the probability for binding 18C6 with proximate salt bridge or hydrogen bonding interactions interfering the most 6 Structural information about the protein is therefore obtained because the local chemical environment surrounding each residue is a function of protein structure 47 In the end a distribution of peaks known as a SNAPP distribution representing the number of 18C6s attached to the protein is observed in the mass spectrometer and is characteristic of a particular protein conformation Changes to the protein structure will typically lead to changes in the SNAPP distribution therefore the strength of SNAPP is in measuring structural differences for either static or dynamic systemsSNAPP is closely related to other mass spectrometry MS based protein structure determination methods such as charge state distribution analysis 8 9 10 hydrogen/deuterium H/D exchange 9 10 11 and covalent labeling 12 but also differs in several important ways H/D exchange monitors protein structure as a function of the exchange of amide hydrogens The overall number locations and rates of exchanges can be used to interrogate structure 13 Various covalent labeling methods can also be used to probe structure although in this case reactions are ideally restricted to a single modification to avoid perturbation of the target structure 14 The primary difference between SNAPP and both of these methods is that the reporting chemistry in SNAPP is highly reversible if present at all in solution and information is only encoded during the later stages of ESI when the protein is transitioning into the gas phase This enables SNAPP to probe highly dynamic systems where structural changes may be occurring rapidly in solution and on the same timescale as H/D exchange or covalent labeling If for example a protein adopted an unfolded and folded state under given conditions the SNAPP distributions for both would be determined independently during ESI and observable in different charge states due to the different sizes as long as the structural transition took longer than a few milliseconds the time required to desolvate the protein SNAPP is therefore an appropriate method to interrogate the structural features of lymphotactin which undergoes structural changes on a significantly longer timescale 2Herein it is demonstrated that the Ltn10 and Ltn40 structures yield easily distinguishable SNAPP distributions The wild type protein yields results that are most similar to the W55D variant The addition of acid or organic denaturants does not have a large impact on the SNAPP distributions for the wild type CC3 or W55D proteins Reduction and blocking of the disulfide bonds affects the structures more dramatically and yields shifts in the SNAPP distributions that are consistent with significant unfolding Data obtained from NMR experiments supports this conclusion The effects of cold temperature on all three proteins are explored Finally a combination of SNAPP and radical chemistry is utilized to compare the gas phase structures of the wild type CC3 and W55D proteinsTo reduce disulfide bonds in the lymphotactin proteins 60 μL solution containing 10 nmol protein and excess amount of DTT 10 mM was incubated at 54 °C for 45 min Free cysteine and excess DTT were treated with a stoichiometric amount of IAA 122 μmol for wild type Ltn and W55D 124 μmol for CC3 to block all free thiol groups The blocked proteins were purified by a protein trap Michrom Bioresources Inc Auburn CA USA lyophilized and stored at –20 °C for subsequent useCC3 wild type Ltn and W55D stock solutions were diluted to 7 μM in H2O respectively The final concentration of 18C6 in SNAPP solutions was 84 μM Mass spectra were obtained using an LTQ linear ion trap mass spectrometer Thermo Fisher Scientific San Jose CA USA equipped with a standard ESIsource Protein samples mixed with 18C6 were directly infused into LTQ mass spectrometer The electrospray parameters such as spray voltage sheath gas flow rates capillary voltage temperature etc were optimized and were similar to parameters described previously for SNAPP experiments 4 Once optimized all the parameters were maintained for all SNAPP experiments presented herein The following are the optimized source parameters for all SNAPP experiments spray voltage 48 kV sheath gas flow rates 11 tube lens 160 V capillary voltage 44 V and capillary temperature 275 °C

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