Controlled activation of protein rotational dynamics using smart hydrogel tethering.

Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications that take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a poly(ethylene glycol) (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with (13)C and (15)N, permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. The protein dynamics is suppressed upon initial formation of hydrogels, with a concomitant increase in protein stability. Relaxation of the hydrogel matrix following transient heating results in enhanced protein dynamics and resolution of substrate-induced large-amplitude domain rearrangements.

Molecular basis of the structural stability of a Top7-based scaffold at extreme pH and temperature conditions.

The development of stable biomolecular scaffolds that can tolerate environmental extremes has considerable potential for industrial and defense-related applications. However, most natural proteins are not sufficiently stable to withstand non-physiological conditions. We have recently engineered the de novo designed Top7 protein to specifically recognize the glycoprotein CD4 by insertion of an eight-residue loop. The engineered variant exhibited remarkable stability under chemical and thermal denaturation conditions. In the present study, far-UV CD spectroscopy and explicit-solvent MD simulations are used to investigate the structural stability of Top7 and the engineered variant under extreme conditions of temperature and pH. Circular dichroism measurements suggest that the engineered variant Top7(CB1), like Top7, retains its structure at high temperatures. Changes in CD spectra suggest that there are minor structural rearrangements between neutral and acidic environments for both proteins but that these do not make the proteins less stable at high temperatures. The anti-parallel beta-sheet is well conserved within the timescale simulated whereas there is a decrease of helical content when low pH and high-temperature conditions are combined. Concerted alanine mutations along the alpha-helices of the engineered Top7 variant did not revert this trend when at pH 2 and 400K. The structural resilience of the anti-parallel beta-sheet suggests that the protein scaffold can accommodate varying sequences. The robustness of the Top7 scaffold under extreme conditions of pH and temperature and its amenability to production in inexpensive bacterial expression systems reveal great potential for novel biotechnological applications.

Engineering an ultra-stable affinity reagent based on Top7.

Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly stable, protein-based affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail. We have engineered Top7--a highly stable, computationally designed protein--to specifically bind human CD4 by inserting a peptide sequence derived from a CD4-specific antibody. Molecular dynamics simulations were used to evaluate the structural effect of the peptide insertion at a specific site within Top7 and suggest that this Top7 variant retains conformational stability over 100 degrees C. This engineered protein specifically binds CD4 and, consistent with simulations, is extremely resistant to thermal and chemical denaturation--retaining its secondary structure up to at least 95 degrees C and requiring 6 M guanidine to completely unfold. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent which could serve as a robust and inexpensive alternative to antibodies.

Different conformational switches underlie the calmodulin-dependent modulation of calcium pumps and channels.

We have used fluorescence spectroscopy to investigate the structure of calmodulin (CaM) bound with CaM-binding sequences of either the plasma membrane Ca-ATPase or the skeletal muscle ryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimide at engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions between these opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformational states of bound CaM. Complementary measurements take advantage of the unique conserved Trp within CaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurements of both a local and global peptide structure. We find that CaM binds with high affinity in a collapsed structure to the CaM-binding sequences of both the Ca-ATPase and RyR1, resulting in excimer formation that is indicative of contact interactions between the N- and the C-domains of CaM in complex with these CaM-binding peptides. There is a 4-fold larger amount of excimer formation for CaM bound to the CaM-binding sequence of the Ca-ATPase in comparison to RyR1, indicating a closer structural coupling between CaM domains in this complex. Prior to CaM association, the CaM-binding sequences of the Ca-ATPase and RyR1 are conformationally disordered. Upon CaM association, the CaM-binding sequence of the Ca-ATPase assumes a highly ordered structure. In comparison, the CaM-binding sequence of RyR1 remains conformationally disordered irrespective of CaM binding. These results suggest an important role for interdomain contact interactions between the opposing domains of CaM in stabilizing the structure of the peptide complex. The substantially different structural responses associated with CaM binding to Ca-ATPase and RyR1 indicates a plasticity in their respective binding mechanisms that accomplishes different physical mechanisms of allosteric regulation, involving either the dissociation of a C-terminal regulatory domain necessary for pump activation or the modulation of intersubunit interactions to diminish RyR1 channel activity.

Loss of the calmodulin-dependent inhibition of the RyR1 calcium release channel upon oxidation of methionines in calmodulin.

The oxidation of methionines in calmodulin (CaM) can affect the activity of calcium pumps and channels to modulate the amplitude and duration of calcium signals. We have therefore investigated the possible oxidation of CaM in skeletal muscle and its effect on the CaM-dependent regulation of the RyR1 calcium release channel. Taking advantage of characteristic reductions in electrophoretic mobility determined by SDS-PAGE, we find that approximately two methionines are oxidized in CaM from skeletal muscle. The functional effect of CaM oxidation on the open probability of the RyR1 calcium release channel was assessed through measurements of [3H]ryanodine binding using a heavy sarcoplasmic reticulum preparation enriched in RyR1. There is a biphasic regulation of RyR1 by unoxidized CaM, in which calcium-activated CaM acts to enhance the calcium sensitivity of channel closure, while apo-CaM functions to enhance channel activity at resting calcium levels. We find that physiological levels of CaM oxidation preferentially weaken the CaM-dependent inhibition of the RyR1 calcium release channel observed at activating micromolar levels of calcium. In contrast, the oxidation of CaM resulted in minimal functional changes in the CaM-dependent activation of RyR1 at resting nanomolar calcium levels. Oxidation does not significantly affect the high-affinity binding of calcium-activated CaM to the CaM-binding sequence of RyR1; rather, methionine oxidation disrupts interdomain interactions between the opposing domains of CaM in complex with the CaM-binding sequence of RyR1 that normally function as part of a conformational switch associated with RyR1 inhibition. These results suggest that the oxidation of CaM can contribute to observed elevations in intracellular calcium levels in response to conditions of oxidative stress observed during biological aging. We suggest that the sensitivity of RyR1 channel activity to CaM oxidation may function as part of an adaptive cellular response that enhances the duration of calcium transients to promote enhanced contractility.

Identification of a denitrase activity against calmodulin in activated macrophages using high-field liquid chromatography--FTICR mass spectrometry.

We have identified a denitrase activity in macrophages that is upregulated following macrophage activation, which is shown by mass spectrometry to recognize nitrotyrosines in the calcium signaling protein calmodulin (CaM). The denitrase activity converts nitrotyrosines to their native tyrosine structure without the formation of any aminotyrosine. Comparable extents of methionine sulfoxide reduction are also observed that are catalyzed by endogenous methionine sulfoxide reductases. Competing with repair processes, oxidized CaM is a substrate for a peptidase activity that results in the selective cleavage of the C-terminal lysine (i.e., Lys148) that is expected to diminish CaM function. Thus, competing repair and peptidase activities define the abundances and functionality of CaM in modulating cellular metabolism in response to oxidative stress, where the presence of the truncated CaM species provides a useful biomarker for the transient appearance of oxidized CaM.

Calcium occupancy of N-terminal sites within calmodulin induces inhibition of the ryanodine receptor calcium release channel.

Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614-N3643) located within the central portion of the primary sequence. However, it is presently unclear whether the identified CaM-binding sequence in association with CaM (a) senses calcium over the physiological range of calcium concentrations associated with RyR1 regulation or alternatively, (b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene)maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association of both apo-CaM and calcium-activated CaM with RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-dependent activation of these individual domains. Fluorescence changes upon calcium activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM at resting calcium levels; at calcium levels associated with muscle contraction, activation of the N-terminal domain occurs with concomitant increases in the fluorescence intensity of PyC-CaM that is associated with structural changes within the CaM-binding sequence of RyR1. Occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 +/- 0.4 microM, suggesting a direct regulation of RyR1 function upon the calcium-dependent activation of CaM. These results indicate that occupancy of the N-terminal domain calcium binding sites in CaM bound to the identified CaM-binding sequence K3614-N3643 induces conformational rearrangements within the complex between CaM and RyR1 responsible for the CaM-dependent modulation of the RyR1 calcium release channel.

Disruption of interdomain interactions via partial calcium occupancy of calmodulin.

Binding of calcium to CaM exposes clefts in both N- and C-domains to promote their cooperative association with a diverse array of target proteins, functioning to relay the calcium signal regulating cellular metabolism. To clarify relationships between the calcium-dependent activation of individual domains and interdomain structural transitions associated with productive binding to target proteins, we have utilized three engineered CaM mutants that were covalently labeled with N-(1-pyrene) maleimide at introduced cysteines in the C- and N-domains, i.e., T110C (PyC-CaM), T34C (PyN-CaM), and T34C/T110C (Py2-CaM). These sites were designed to detect known conformers of CaM such that upon association with classical CaM-binding sequences, the pyrenes in Py2-CaM are brought close together, resulting in excimer formation. Complementary measurements of calcium-dependent enhancements of monomer fluorescence of PyC-CaM and PyN-CaM permit a determination of the calcium-dependent activation of individual domains and indicate the sequential calcium occupancy of the C- and N-terminal domains, with full saturation at 7.0 and 300 microM calcium, respectively. Substantial amounts of excimer formation are observed for apo-CaM prior to peptide association, indicating that interdomain interactions occur in solution. Calcium binding results in a large and highly cooperative reduction in the level of excimer formation; its calcium dependence coincides with the occupancy of C-terminal sites. These results indicate that interdomain interactions between the opposing domains of CaM occur in solution and that the occupancy of C-terminal calcium binding sites is necessary for the structural coupling between the opposing domains associated with the stabilization of the interdomain linker to enhance target protein binding.

Essential role for Pro21 in phospholamban for optimal inhibition of the Ca-ATPase.

We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP(21), which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attain optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro(21) was mutated to an alanine (P21A-PLB(C)); in this construct, all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB, and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca(1/2)) of the Ca-ATPase, 290 +/- 10 nM, is shifted to 580 +/- 20 nM when co-reconstituted with PLB(C) (Pro21) as a result of a reduction in the cooperativity associated with the calcium-dependent structural transition. Kinetic simulations indicate that PLB(C) association with the Ca-ATPase results in a 75% reduction in the equilibrium constant associated with the formation of the second high-affinity calcium binding site. In comparison, there is a 43% reduction in KCa(1/2) upon reconstitution of the Ca-ATPase with P21A-PLB(C), which can be simulated by decreasing the equilibrium constant associated with the calcium-dependent structural activation by 50%. The diminished inhibitory action of P21A-PLB(C) is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the alpha-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLB(C) are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLB(C) that result in a reduction in the ability of the amino-terminal cytosolic portion of PLB to productively inhibit the Ca-ATPase. Further, these results suggest that the unstructured characteristics of the flexible hinge region in PLB are critical for optimal inhibitory interactions with the Ca-ATPase and suggest its role as a conformational switch.