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Abstract Background Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers. Methods Mice carrying human (FaDudd) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared. Results In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDudd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker. Conclusions The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.

Calmodulin (CaM) allosterically regulates the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1): apo CaM activates the channel, while (Ca(2+))4-CaM inhibits it. CaM-binding RyR1 residues 1975-1999 and 3614-3643 were proposed to allow CaM to bridge adjacent RyR1 subunits. Fluorescence anisotropy titrations monitored the binding of CaM and its domains to peptides encompassing hRyR(11975-1999) or hRyR1(3614-3643). Both CaM and its C-domain associated in a calcium-independent manner with hRyR1(3614-3643) while N-domain required calcium and bound ~250-fold more weakly. Association with hRyR1(11975-1999) was weak. Both hRyR1 peptides increased the calcium-binding affinity of both CaM domains, while maintaining differences between them. These energetics support the CaM C-domain association with hRyR1(3614-3643) at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR(11975-1999) alone was insufficient to support CaM bridging adjacent RyR1 subunits. Other proteins or elements of the hRyR1 structure must contribute to the energetics of CaM-mediated regulation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Calmodulin (CaM) is the primary transducer of calcium fluxes in eukaryotic cells. Its two domains allosterically regulate myriad target proteins through calcium-linked association and conformational change. Many of these proteins have a basic amphipathic alpha-helix (BAA) motif that binds one or both CaM domains. Previously, we demonstrated domain-specific binding of melittin, a model BAA peptide, to Paramecium CaM (PCaM): C-domain mutations altered the interaction with melittin, whereas N-domain mutations had no discernable effect. Here, we report on the use of fluorescence and NMR spectroscopy to measure the domain-specific association of melittin with calcium-saturated ((Ca(2+))(4)-PCaM) or calcium-depleted (apo) PCaM, which has enabled us to determine the free energies of calcium binding to the PCaM-melittin complex, and to estimate interdomain cooperativity. Under apo conditions, melittin associated with each PCaM domain fragment (PCaM(1-80) and PCaM(76-148)), as well as with the C-domain of full-length PCaM (PCaM(1-148)). In the presence of calcium, all of these interactions were again observed, in addition to which an association with the N-domain of (Ca(2+))(4)-PCaM(1-148) occurred. This new association was made possible by the fact that melittin changed the calcium-binding preferences for the domains from sequential (C > N) to concomitant, decreasing the median ligand activity of calcium toward the N-domain 10-fold more than that observed for the C-domain. This selectivity may be explained by a free energy of cooperativity of -3 kcal/mol between the N- and C-domains. This study demonstrates multiple domain-selective differences in the interactions between melittin and PCaM. Our findings support a model that may apply more generally to ion channels that associate with the C-domain of CaM under low (resting) calcium conditions, but rearrange when calcium binding triggers an association of the N- domain with the channel., ((c) 2008 Wiley-Liss, Inc.)

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.

Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 A) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875-898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent exposed. NR1 F880 filled the CaM C-domain pocket, whereas T886 was closest to the N-domain pocket. This 1-7 pattern was most similar to that in the CaM-MARCKS complex. Comparison of CaM-ligand wrap-around conformations identified a core tetrad of CaM C-domain residues (FLMM(C)) that contacted all ligands consistently. An identical tetrad of N-domain residues (FLMM(N)) made variable sets of contacts with ligands. This CaM-NR1C1 structure provides a foundation for designing mutants to test the role of CaM in NR1 trafficking as well as insights into how the homologous CaM domains have different roles in molecular recognition.

Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.

Binding of the transcription factor PU.1 to its DNA binding motif regulates the expression of a number of B-cell- and myeloid-specific genes. The long terminal repeat (LTR) of macrophage-tropic strains of equine infectious anemia virus (EIAV) contains three PU.1 binding sites, namely an invariant promoter-proximal site as well as two upstream sites. We have previously shown that these sites are important for EIAV LTR activity in primary macrophages (W. Maury, J. Virol. 68:6270-6279, 1994). Since the sequences present in these three binding motifs are not identical, we sought to determine the role of these three sites in EIAV LTR activity. While DNase I footprinting studies indicated that all three sites within the enhancer were bound by recombinant PU.1, reporter gene assays demonstrated that the middle motif was most important for basal levels of LTR activity in macrophages and that the 5' motif had little impact. The impact of the 3' site became evident in Tat transactivation studies, in which the loss of the site reduced Tat-transactivated expression 40-fold. In contrast, elimination of the 5' site had no effect on Tat-mediated activity. Binding studies were performed to determine whether differences in PU.1 binding affinity for the three sites correlated with the relative impact of each site on LTR transcription. While small differences were observed in the binding affinities of the three sites, with the promoter-proximal site having the strongest binding affinity, these differences could not account for the dramatic differences observed in the transcriptional effects. Instead, the promoter-proximal position of the 3' motif appeared to be critical for its transcriptional impact and suggested that the PU.1 sites may serve different roles depending upon the location of the sites within the enhancer. Infectivity studies demonstrated that an LTR containing an enhancer composed of the three PU.1 sites was not sufficient to drive viral replication in macrophages. These findings indicate that while the promoter-proximal PU.1 site is the most critical site for EIAV LTR activity in the presence of Tat, other elements within the enhancer are needed for EIAV replication in macrophages.

Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1-75), the addition of linker residues 76 to 80 (MKEQD) raised the Tm by 9 degrees C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., [Biochemistry 2002;41:15-20]), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1-74 to PCaM1-80). For the first 4, PCaM1-74 through PCaM1-77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5 degrees C in Tm and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1-77 through PCaM1-80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of 15N-PCaM1-74 and 15N-PCaM1-80 and analysis of high-resolution structural models suggest these residues are latched to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains., (Copyright 2003 Wiley-Liss, Inc.)

Calmodulin (CaM) is an intracellular calcium-binding protein essential for many pathways in eukaryotic signal transduction. Although a structure of Ca(2+)-saturated Paramecium CaM at 1.0 A resolution (1EXR.pdb) provides the highest level of detail about side-chain orientations in CaM, information about an end state alone cannot explain driving forces for the transitions that occur during Ca(2+)-induced conformational switching and why the two domains of CaM are saturated sequentially rather than simultaneously. Recent studies focus attention on the contributions of interdomain linker residues. Electron paramagnetic resonance showed that Ca(2+)-induced structural stabilization of residues 76-81 modulates domain coupling [Qin and Squier (2001) Biophys. J. 81, 2908-2918]. Studies of N-domain fragments of Paramecium CaM showed that residues 76-80 increased thermostability of the N-domain but lowered the Ca(2+) affinity of sites I and II [Sorensen et al. (2002) Biochemistry 41, 15-20]. To probe domain coupling during Ca(2+) binding, we have used (1)H-(15)N HSQC NMR to monitor more than 40 residues in Paramecium CaM. The titrations demonstrated that residues Glu78 to Glu84 (in the linker and cap of helix E) underwent sequential phases of conformational change. Initially, they changed in volume (slow exchange) as sites III and IV titrated, and subsequently, they changed in frequency (fast exchange) as sites I and II titrated. These studies provide evidence for Ca(2+)-dependent communication between the domains, demonstrating that spatially distant residues respond to Ca(2+) binding at sites I and II in the N-domain of CaM.

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition.

A hydrophobic core is a widely accepted determinant of protein stability. However, regulatory proteins undergoing ligand-induced conformational switching may expose interior residues to solvent and cannot afford to be extremely rigid. Optimizing the energetic balance between stability and binding is challenging. The addition of five interdomain residues to rat and Paramecium calmodulin N-domain fragments (residues 1-75) increased their thermostability by 9 degrees C and lowered their calcium affinity by a factor of 4. This demonstrates that the flexible linker regulates functional properties as well as tethering the neighboring domains and that protein stability may be increased markedly by minor modifications of the C-terminus. The sensitivity of this domain to few and conservative variations in helices A and D (D2E, S17A, T70S and M71L) is demonstrated by the rat CaM fragments having lower stability and higher calcium affinity than fragments of the same length derived from Paramecium CaM.

Calmodulin (CaM) is a small acidic protein essential to calcium-mediated signal transduction. Conformational change driven by calcium binding controls its selective activation of myriad target proteins. In most well characterized cases, both homologous domains of CaM interact with a target protein. However, physiologically separable roles for the two domains were demonstrated by mutants of Paramecium tetraurelia [Kung, C. et al. (1992) Cell Calcium 13, 413], some of which have altered calcium affinities [Jaren, O. R. et al. (2000) Biochemistry 39, 6881]. To determine whether these mutants can associate with canonical targets in a calcium-dependent manner, their ability to bind melittin was assessed using analytical gel permeation chromatography, analytical ultracentrifugation, and fluorescence spectroscopy. The Stokes radius of wild-type PCaM and 11 of the mutants decreased dramatically upon binding melittin in the presence of calcium. Fluorescence spectra and sedimentation velocity studies showed that melittin bound to wild-type PCaM and mutants in a calcium-independent manner. However, there were domain-specific perturbations. Mutations in the N-domain of PCaM did not affect the spectrum of melittin (residue W19) under apo or calcium-saturated conditions, whereas most of the mutations in the C-domain did. These data are consistent with a calcium-dependent model of sequential target association whereby melittin (i) binds to the C-domain of PCaM in the absence of calcium, (ii) remains associated with the C-domain upon calcium binding to sites III and IV, and (iii) subsequently binds to the N-domain upon calcium binding to sites I and II of CaM, causing tertiary collapse.

To dissect the chemical basis for interactions controlling regulatory properties of macromolecular assemblies, it is essential to explore experimentally the linkage between ligand binding, conformational change, and subunit assembly. There are many advantages to using techniques that will probe the occupancy of individual binding sites or monitor conformational responses of individual residues, as described here. Proteolytic footprinting titrations may be used to infer binding free energies for ligands interacting with multiple sites or domains and to detect otherwise unrecognized "silent" interdomain interactions. Microgram quantities of pure protein are required, which is low relative to the hundreds of milligrams needed for comparable discontinuous equilibirum titrations monitored by NMR. By running comparative studies with several proteases, it is easy to determine whether resulting titration curves are consistent, independent of the protease used and therefore representative of the structural response of the protein to ligand binding or other differences in solution conditions (pH, salt, temperature). The results from multiple techniques (e.g., NMR, fluorescence, and footprinting) applied to aliquots from the same discontinuous titration may be compared easily to test for consistency. Classic methods for determining thermodynamic and kinetic properties of calcium binding to calmodulin include filter binding and equilibrium or flow dialysis (employing the isotope 45Ca), spectroscopic studies of stopped-flow fluorescence, calorimetry, and direct ion titrations. A cautionary note is that many different sets of microscopic data would be consistent with a single set of macroscopic constants determined by classic methods. This was well illustrated in Fig. 9. Thus, while it is important to compare results with those obtained by classic binding methods, they are, by definition, incapable of resolving the microscopic constants of interest. Thus, there is only one "direction" for comparison. Quantitative proteolytic footprinting titrations applied to studying calmodulin provided the first direct quantitative estimate of negative interactions between domains. Although studies of site-knockout mutants had suggested interactions between domains, this approach gave the first evidence for the pathway of anticooperative interactions between domains by showing that helix B responds structurally to calcium binding to sites III and IV in the C-domain. Despite two decades of study of calmodulin and the application of limited proteolysis studies to the apo and fully saturated forms, this finding emerged only when titration studies were undertaken as described. This highlights the general observation that while the behavior of the intermediate states in a cooperative switch are the key elements of the transition mechanism, they are the most difficult to observe. The unexpected finding that the isolated domains are nearly equivalent in their calcium-binding properties (Fig. 23 B) leaves us with many of the questions we had at the start: How does the sum of two nearly equivalent domains result in a molecule that switches sequentially rather than simultaneously? But it underscores why it is not yet possible to understand similar proteins by sequence gazing alone.

Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) protein that is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotes examined. All homologues are heterotrimeric complexes with subunits of approximately 70, approximately 32, and approximately 14 kDa. While RPA homologues are evolutionarily conserved, they are not functionally equivalent. To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and low cooperativity. However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing the high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties. In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weak ssDNA binding activity. However, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. We conclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA. These data also indicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins.

Calmodulin (CaM) is an essential protein that exerts exquisite spatial and temporal control over diverse eukaryotic processes. Although the two half-molecule domains of CaM each have two EF-hands and bind two calcium ions cooperatively, they have distinct roles in activation of some targets. Interdomain interactions may mediate coordination of their actions. Proteolytic footprinting titrations of CaM [Pedigo and Shea (1995) Biochemistry 34, 1179-1196; Shea, Verhoeven, and Pedigo (1996) Biochemistry 35, 2943-2957] showed that calcium binding to the high-affinity sites (III and IV in the C-domain) alters the conformation of helix B in the N-domain despite sites I and II being vacant. This may arise from calcium-induced disruption of interactions between the apo domains. In this study, comparing the cloned domains (residues 1-75, 76-148) to whole CaM, the proteolytic susceptibility of helix B in the apo isolated N-domain was higher than in apo CaM. The isolated N-domain was monotonically protected by calcium binding and had a higher calcium affinity than when part of whole CaM. The change in affinity was small (1-1.5 kcal/mol) but acted to separate the domain saturation curves of whole CaM. Unfolding enthalpies and melting temperatures of the apo isolated domains did not correspond to the two transitions resolved for apo CaM. In summary, the interactions between domains of apo CaM protected the N-domain from proteolysis and raised its Tm by 10 degrees C, demonstrating that CaM is not the sum of its parts.

Calmodulin (CaM) is an intracellular cooperative calcium-binding protein essential for activating many diverse target proteins. Biophysical studies of the calcium-induced conformational changes of CaM disagree on the structure of the linker between domains and possible orientations of the domains. Molecular dynamics studies have predicted that Ca4(2+)CaM is in equilibrium between an extended and compact conformation and that Arg74 and Arg90 are critical to the compaction process. In this study gel permeation chromatography was used to resolve calcium-induced changes in the hydrated shape of CaM at pH 7.4 and 5.6. Results showed that mutation of Arg 74 to Ala increases the R(s) as predicted; however, the average separation of domains in Ca4(2+)-CaM was larger than predicted by molecular dynamics. Mutation of Arg90 to Ala or Gly affected the dimensions of apo-CaM more than those of Ca4(2+)-CaM. Calcium binding to CaM and mutants (R74A-CaM, R90A-CaM, and R90G-CaM) lowered the Stokes radius (R(s)). Differences between R(s) values reported here and Rg values determined by small-angle x-ray scattering studies illustrate the importance of using multiple techniques to explore the solution properties of a flexible protein such as CaM.