Conformational changes of recombinant Ca²⁺–ATPase studied by reaction‐induced infrared difference spectroscopy

Abstract

Recombinant Ca2+ -ATPase was expressed in Saccharomyces cerevisiae with a biotin-acceptor domain linked to its C-terminus by a thrombin cleavage site. We obtained 200 lg of~70% pure recombinant sarcoendoplasmic reticulum Ca 2+ -ATPase isoform 1a (SERCA1a) from a 6-L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca 2 E1P and Ca 2 E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca 2 E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.

Horse gluteus is a null-sarcolipin muscle with enhanced sarcoplasmic reticulum calcium transport

“…We utilized quantitative immunoblotting and Coomassie densitometry to compare the relative amount of SERCA protein in SR vesicles from horse versus rabbit muscle. For standardization, the SERCA content of SR vesicles from rabbit fast-twitch (white) muscle has been determined to be 55-70% of total protein (weight/weight), i.e., 5.0-6.4 nmol SERCA/mg rabbit SR protein (98,(100)(101)(102). Immunoblotting with anti-SERCA1 mAb VE12 1 G9 demonstrated that SR vesicles from horse gluteal muscle express ~35% the relative amount of SERCA1 as compared to rabbit SR (Figure 5A, Figure S5), although this is probably a slight underestimate of total SERCA content, since horse SR (10KP) also expresses a small amount of ATP2A2 (SERCA2) (Figure 1), which is incompatible with the use of anti-SERCA1 mAb VE12 1 G9.…”

“…Semi-quantitative immunoblot analysis of four horse SR preps using pAb GS3379 and a synthetic horse SLN standard determined that native SLN is expressed at 0.16 ± 0.015 nmol SLN/mg SR protein (Figure S6). The expression level of SERCA in horse SR is estimated at 2.7 nmol SERCA/mg SR protein (see above), per content assessed by Coomassie densitometry (Figure S4) and SERCA immunoblotting (Figure 5, Figure S5) using rabbit SR as a relative SERCA standard, with 5.0-6.4 nmol SERCA/mg SR protein (98,100,101). Thus, quantitative immunoblotting using the anti-horse-SLN pAb 3379 and a synthetic horse SLN peptide standard determined that SR vesicles from horse gluteal muscle express a minor ratio of 0.059 ± 0.005 SLN/SERCA (mol/mol protein).…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…In the case of membrane proteins and photoactive proteins, a variety of systems have been investigated using the approaches described in this review and first introduced for bacteriorhodopsin. A few examples are the photosystem II [116,152], photoactive yellow protein (PYP) [59,118,124,246]; Ca 2+ -ATPase [21,22,52,98,133]; green fluorescent protein (GFP) and potassium channels [92,230]. One measure of the growth of FTIR difference spectroscopy of biomolecules is shown in Fig.…”

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

“…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 2+ ATPase transporter has been characterized in a purified form of the full‐length protein from a recombinant source using FTIR spectroscopy . Therefore, optimization of triggering conditions in non‐native environments aimed at proteins from recombinant sources will significantly increase the number of targets for time‐resolved studies.…”

“…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. [28] Therefore, optimization of triggering conditions in non-native environments aimed at proteins from recombinant sources will significantly increase the number of targets for time-resolved studies. In a first effort to track the reaction of Type-I ATPases, hydrolysis of ATP was monitored for a Cu 1 ATPase construct containing only the soluble nucleotide-binding and phosphorylation domains using timeresolved FTIR spectroscopy.…”