Application of a New Immobilization H/D Exchange Protocol: A Calmodulin Study

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1 Application of a New Immobilization H/D Exchange Protocol: A Calmodulin Study Jiang Zhao; Mei Zhu; Daryl E. Gilblin; Michael L. Gross Washington University Center for Biomrdical and Bioorganic Mass Spectrometry: A NIH-Supported Resource Center, Department of Chemistry, Washington University, St. Louis, MO (

2 Overview 1. Purpose: Develop a novel H/D exchange protocol and apply it to examine the degree of conformational changes in calmodulin induced by Ca 2+ binding 2. Methods: A new immobilization procedure for protein in H/D exchange was coupled online with ESI mass spectrometry 3. Results: The new H/D exchange protocol improves both sensitivity and mass resolving power, allowing H/D exchange experiments at protein concentrations of.5 mm. A two-step process for calmodulin/calcium binding is observed by applying the new protocol.

3 1.Calmodulin: ( Fig. 1 ) A small, acidic protein of 148 AA, ~ 17 kda Introduction Contains a central helix, two globular domains and four Ca 2+ - binding sites ServesasCa 2+ -dependent regulator in many metabolic pathways Although the binding of Ca 2+ to calmodulin and the induced conformational changes in the protein have been widely studied, many important details remain to be resolved. H/D exchange followed by MS is one approach

4 2. Probing conformational changes in calmodulin using a new H/D exchange protocol: H/D exchange in combination with ESI MS is a promising tool to probe higher order of protein structure and function. A novel H/D exchange protocol is reported here, allowing high exchange ratio in both forward and back directions, and in-situ on-column quenching and desalting. Building on our report [ JASMS, 1, 711 (1999) ], we applied the new H/D exchange methodology to calmodulin and its conformational changes induced by calcium binding.

5 Methods 1. Materials: Calcium-free porcine calmodulin ( CaM : MW 1679 Da) was obtained from Ocean Biologics Co., Edmonds, WA. Wild-type paramecium calmodulin ( PCaM : MW Da) was donated by Dr. Madeline A. Shea in the Univ. of Iowa. 2. Instrumentation: HPLC was carried out using a Waters 6MS pump with an isocratic flow of 18% H 2 O, 2% acetic acid and 8% CH 3 CN. The flow, split by a LC-packing splitter at 1:1, was introduced into a Microtech Zorbax C-18 SBW column (1 mm x.32 mm). ESI MS was done with a Finnigan LCQ in both full scan and multiple zoom-scan modes. 3. Sample Preparation The CaM stock solution (e.g.: 1 mm) was prepared by

6 dissolving 1 mg protein in 59.6 ml.4 M NH 4 OAc buffer (ph 7, adjusted with NH 4 OH). For the titration, 2 ml aliquots of protein were taken and incubated with 2 ml of Ca(OAc) 2 solution at various concentrations at room temperature for 3 min. 4. Immobilization H/D exchange protocol (Fig. 2) After allowing protein to interact with Ca 2+ in aqueous buffer, D 2 O was added to initiate exchange at neutral ph (D 2 O/H 2 O = 99/1). The exchange solution was then injected onto a small C-18 column, which was cooled to C. The protein, concentrated and immobilized on the column, was washed with chilled formic acid solution (ph 2.5) to quench the uptake of deuterium, back-exchange the facile side-chain H s ( H 2 O/D 2 O > 1/1), and remove salts. An isocratic flow of solvent with high organic composition was used to rapidly elute the protein and submit it to ESI-MS.

7 1x D 2 O Fig.2: Immobilization Assay Waste Mass analyzer Waste Mass analyzer Loading 1~2 ul Injector Solvent pump Injector Solvent pump Waste Mass analyzer Washing Back exchange Eluting Waste Mass analyzer Injector Solvent pump Injector Solvent pump

8 Results and Discussion 1. The new H/D exchange protocol greatly improves both sensitivity and resolving power in ESI-MS analysis The new H/D exchange method showed several significant improvements over the previous protocol reported recently [JASMS, 1999, 711]. The improvements are: (a) Better sensitivity (Figs 3-5 ) Since metal cations are removed prior to MS analysis, protein from various binding states all reverse back to its apo-state, signal-to-noise ratio is improved. Because the protein is concentrated after the forward exchange, lower amounts and concentrations are needed. Therefore, it is possible to analyze less water-soluble or low conc. of proteins.

9 Fig. 5 shows successful H/D exchange at [CaM] = [PCaM] =.5 mm. By injecting larger volume of exchange solution on column, we obtained both ESI spectra of similar quality and H/D exchange kinetics of two calmodulins. Paramecium PCaM exchanges more slowly and to a lower extent than porcine CaM. (b) Higher resolving power A high exchange ratio was maintained in both H/D exchange directions so that there was a lower isotope distribution. When multiple zoom scan mode was engaged, even higher resolving was achieved. (Fig. 4) Much less metal ion interference was observed.

10 1. Calcium Titration With the improved protocol, we re-examined the Ca 2+ titration of calmodulin. All data for the titration were measured immediately after H/D exchange-in at room temperature for 1 h. With the addition of Ca 2+ increments, calmodulin undergoes less H/D exchange, indicating formation of Ca 2+ -bounded calmodulin species, which have tighter, less solvent-accessible structures than apo-calmodulin. The resulting titration curve for [CaM] = 15 mm ( Fig. 6 ) is similar in shape and in the location of the global minimum ([Ca 2+ ] total =.25 mm; CaM-4Ca > 85% of all forms of Ca 2+ -bound calmodulin) to the one previously reported.

11 We also resolved a second minimum at.4 mm of Ca 2+, which may correspond to CaM complexed with two Ca 2+, suggesting that CaM binds to Ca 2+ by a two-step process, a hypothesis that is supported by the fractional species calculations ( Fig. 6 lower panel ). The titration curve for [CaM] = 5 mm ( Fig. 7 ) also agreed with this observation. The second minimum shifted to [Ca 2+ ] total =.2 mm, which corresponds to CaM-2Ca as the predominant species according to the fractional species calculation at [CaM] = 5 mm ( Fig. 7 lower panel ). The absolute number of amide hydrogens exchanged in the two titration curves did not correlate well, possibly due to temperature variations between the two experiments and to the use of lower flow rates for the 5-mM experiment (allowing for more back exchange).

12 Conclusions The novel Immobilization H/D Exchange Protocol improves greatly the sensitivity and mass resolving power of ESI-MS. The new protocol enables the study of proteins that are only available in small quantity or low concentrations (e.g..5 mm). A two-step calmodulin calcium binding process is observed when the new H/D protocol is used for the titration of CaM with Ca 2+. Acknowledgement This work was supported by the National Center for Research Resources of the NIH (grant No. P41RR954). We thank Dr. Madeline A. Shea for providing paramecium Calmodulin.

13 Fig. 3A : Comparison of two H/D exchange protocols --- Typical original full scan ESI mass spectra Old H/D Protocol Forward exchange ratio: 4: Back exchange < 4: New immobilization H/D exchange protocol Forward exchange ratio: 99:1 Back exchange ratio: > 1: M/Z

14 Fig. 3B : Comparison of two H/D exchange protocols --- Typical peak shapes after deconvolution 1 8 Old H/D protocol Forward exchange ratio: 4:1 Back exchange < 4:1 6 FWHH = 5 ~ 1 u New immobilization H/D exchange protocol FWHH=6~2 u Forward exchange ratio: 99:1 Back exchange ratio: > 1: M/Z 174

15 Fig. 4 : Typical full scan and multiple zoom- scan mass spectra before and after deconvolution from using the new protocol Full Scan Full Scan After Deconvolution Multiple Zoom Scan Multiple Zoom Scan After Deconvolution M/Z

16 Fig. 5: H/D Exchange Kinetics Data For CaM and PCaM ([CaM] = [PCaM] =.5 um; RT = 21 Degree Centigrade) Number of Hydrogens Exchanged CaM PCaM Exchange-in Time (min)

17 Fig. 6: H/D Exchange Data For CaM Titration With Ca2+ ( [CaM] = 15 um; RT = 25 Degree Centigrade Number of Hydrogens Exchanged in 1 Hr Calmodulin Binding Fractions vs Total Calcium 1. Fractional Abundance TotalConcentrationofCa2+(mM) Fraction Ca Fraction 1 Ca Fraction 2 Ca Fraction 3 Ca Fraction 4 Ca

18 Fig. 7: H/D Exchange Data For CaM Titration With Ca2+ ( [CaM] = 5 um; RT = 21 Degree Centigrade Number of Hydrogens Exchanged in 1 Hr Calmodulin Binding Fractions vs Total Calcium 1. Fractional Abundance Fraction Ca Fraction 1 Ca Fraction 2 Ca Fraction 3 Ca Fraction 4 Ca Total Concentration of Ca2+ ( mm )

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