Monitoring of secondary and tertiary structure changes in the gastric H⁺/K⁺‐ATPase by infrared spectroscopy

Abstract

Conformational changes occurring in the catalytic cycle of the H 1 /K 1 -ATPase were monitored by Fourier transform infrared spectroscopy (FTIR). Caged compounds were used to release ATP, in the presence of Ca 21 , to induce the transition between the E1 and E1-P conformation of the H 1 /K 1 -ATPase. In addition to bands associated with the photolysis of the caged compounds, some peaks of the difference infrared spectra were associated with changes in secondary structure and modifications of the ionization in the side chains of amino-acid residues (Glu or Asp). These changes were specific to the reaction between the ligand and the enzyme. We estimated that 39 amino acids changed their secondary structure during the reaction and four amino-acid residues were deprotonated. Similar spectral changes appeared when ADP was released from its precursor. The release of P i from the same caged molecule did not induce similar changes. Changes in tertiary structure occurring during the binding of adenosine and phosphorylation of the enzyme were demonstrated by recording hydrogen/deuterium exchange kinetics by attenuated total reflectance FTIR spectroscopy (ATR-FTIR). At least 129 amide protons were involved in a tertiary structure change induced by ATP. This suggested that secondary structure change transduced a much larger tertiary structure modification.Keywords: H 1 /K 1 -ATPase; caged-compounds; catalytic cycle; ATR-FTIR spectroscopy.The gastric H 1 /K 1 -ATPase is the electroneutral ion pump responsible for acid secretion in the gastric mucosa. In this process, cytosolic H 1 is exchanged for extracellular K 1 . The gastric H 1 /K 1 -ATPase is a member of the P-ATPase family and shares many features, including sequence homologies and enzymatic mechanisms with other members such as the Ca 21 -ATPase [1], and especially the Na 1 /K 1 -ATPase [2]. Enzymes of this large family are characterized by the formation of a covalent aspartylphosphate group during their catalytic cycle and the transition between two major conformations called E1 and E2.From a structural point of view, this enzyme is composed of a catalytic a subunit (114 kDa) and a glycosylated b subunit (33 kDa). The a subunit, a membrane multispanning polypeptide, is responsible for the coupling of ATP hydrolysis with ion transport across the membrane. The b subunit, a single membrane spanning peptide, is required for the proper assembly and targeting of the entire protein. The a subunit of the H 1 /K 1 -ATPase consists of 1034 amino-acid residues [2]. It contains the phosphorylation site (Asp385) and Lys517, which is preferentially labeled by fluorescein 5 H -isothiocyanate (FITC). The models based on hydrophobicity profiles and different experimental results predict 10 membrane spanning segments for the a subunit. Although segments M9 and M10 have never been isolated after proteolysis, genetics experiments support this view [2,3]; it is also consistent with the recently published structure of the Ca 21 -ATPase [4]. The b subunit consists of 290 amino-acid residu...

Hydrogen‐deuterium exchange in membrane proteins monitored by IR spectroscopy: A new tool to resolve protein structure and dynamics

“…These results indicate that while 516 residues belong to the fast exchanging class ( k =0.5 min −1 ) in the absence of ligand, only 185 remain in this class in the presence of 2 m M Ca‐ATP. This huge difference can only be explained in term of domain–domain interactions since the secondary structure of the protein does not change significantly in these conditions 14…”

“…1 H/ 2 H exchange kinetics has also been widely used in the field of membrane proteins. It allows the characterization of the protein stability in different experimental conditions5–13 and of conformational changes relevant to enzyme activity 14–17. Recently, it has been shown that combining linear dichroism with 1 H/ 2 H exchange measurement allows the exchange rate to be recorded specifically for the oriented secondary structures in a membrane‐embedded protein 18.…”

Probing the activity of a recombinant Zn2+‐transporting P‐type ATPase

“…The resulting absorbance bands showed both similarities to those observed for Ca 2+ ATPase as well as completely unique spectral features. Similarly, another member of the P‐type family, the H + ,K + ATPase, which is responsible for acidifying the stomach, was isolated from hog gastric fundus and showed unique FTIR spectral features upon activation by caged ATP, although the major inward‐to‐outward facing transition (E1‐to‐E2) was not investigated …”

“…Similarly, another member of the P-type family, the H 1 ,K 1 ATPase, which is responsible for acidifying the stomach, [26] was isolated from hog gastric fundus and showed unique FTIR spectral features upon activation by caged ATP, although the major inward-to-outward facing transition (E1-to-E2) was not investigated. [27] Thus, while a few members of the P-type ATPase family occur at high enough concentration in the membrane to allow characterization by triggering in the native environment, the vast majority would have to be expressed from recombinant sources. For the P-type ATPases, only the Ca 21 ATPase transporter has been characterized in a purified form of the full-length protein from a recombinant source using FTIR spectroscopy.…”

Obtaining information on protein dynamics using FT-IR spectroscopy

“…The time from the reconstitution of lyophilized protein to acquisition of the first spectrum was <60 s. Normally the spectra need to be recorded at 1min intervals during the first 10 min, but the interval for the kinetic scanning is changed to 5 min between 15-90 min and 10 min after 90 min. The total collection time for all spectra varied between different proteins depending on exchange rates (e.g., 3 h for lactose permease, > 48 h for bacteriorhodopsin) 61,62 . During data acquisition, the spectrum for each individual scan is displayed on the page.…”