Jaume Canaves - Academia.edu (original) (raw)

Papers by Jaume Canaves

Research paper thumbnail of Isoform Specific Differences in Binding of a Dual-Specificity A-Kinase Anchoring Protein to Type I and Type II Regulatory Subunits of PKA †

Biochemistry, 2003

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent... more Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIR than to RIR. The higher affinity for RIIR was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIR and RIIR was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIR was independent of solution ionic strength, whereas RIIR had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIR but not to RIIR. These isoform-specific differences provide a foundation for developing isoformspecific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring. † This work was funded by an NIH grant (DK-54441) to S.S.T. L.L.B. was funded by an NIH training grant (5T32 DK-07233).

Research paper thumbnail of Protein Biophysical Properties that Correlate with Crystallization Success in Thermotoga maritima: Maximum Clustering Strategy for Structural Genomics

Journal of Molecular Biology, 2004

Cost and time reduction are two of the driving forces in the development of new strategies for pr... more Cost and time reduction are two of the driving forces in the development of new strategies for protein crystallization and subsequent structure determination. Here, we report the analysis of the Thermotoga maritima proteome, in which we compare the proteins that were successfully expressed, purified and crystallized versus the rest of the proteome. This set of almost 500 proteins represents one of the largest, internally consistent, protein expression and crystallization datasets available. The analysis shows that individual parameters, such as isoelectric point, sequence length, average hydropathy, low complexity regions (SEG), and combinations of these biophysical properties for crystallized proteins define a distinct subset of the T. maritima proteome. The distribution profiles of the various biophysical properties in the expression/crystallization set are then used to extract rules to improve target selection and improve the efficiency and output of structural genomics, as well as general structural biology efforts.

Research paper thumbnail of The Structure of a Eukaryotic Nicotinic Acid Phosphoribosyltransferase Reveals Structural Heterogeneity among Type II PRTases

Structure, 2005

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for cellular redox reactions and... more Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for cellular redox reactions and can act as an important substrate in numerous biological processes. As a result, nature has evolved multiple biosynthetic pathways to meet this high chemical demand. In Saccharomyces cerevisiae, the NAD salvage pathway relies on the activity of nicotinic acid phosphoribosyltransferase (NAPRTase), a member of the phosphoribosyltransferase (PRTase) superfamily. Here, we report the structure of a eukaryotic (yeast) NAPRTase at 1.75 A resolution (locus name: YOR209C, gene name: NPT1). The structure reveals a two-domain fold that resembles the architecture of quinolinic acid phosphoribosyltransferases (QAPRTases), but with completely different dispositions that provide evidence for structural heterogeneity among the Type II PRTases. The identification of a third domain in NAPRTases provides a structural basis and possible mechanism for the functional modulation of this family of enzymes by ATP.

Research paper thumbnail of Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

European Biophysics Journal With Biophysics Letters, 2004

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking ... more Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N2 showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.

Research paper thumbnail of Shotgun crystallization strategy for structural genomics: An Optimized two-tiered crystallization screen against the thermotogamaritima proteome

Acta Crystallographica Section D-biological Crystallography, 2003

Research paper thumbnail of High-throughput Protein Production for X-ray Crystallography and Use of Size Exclusion Chromatography to Validate or Refute Computational Biological Unit Predictions

Journal of Structural and Functional Genomics, 2005

The production of large numbers of highly purified proteins for X-ray crystallography is a signif... more The production of large numbers of highly purified proteins for X-ray crystallography is a significant bottleneck in structural genomics. At the Joint Center for Structural Genomics (JCSG; http://www.jcsg.org), specific automated protein expression, purification, and analytical methods are being utilized to study the proteome of Thermotoga maritima. Anion exchange and size exclusion chromatography (SEC), intended for the production of highly purified proteins, have been automated and the procedures are described here in detail. Analytical SEC has been included as a standard quality control test. A biological unit (BU) is the macromolecule that has been proven or is presumed to be functional. Correct assignment of BUs from protein structures can be difficult. BU predictions obtained via the Protein Quaternary Structure file server (PQS; http://pqs.ebi.ac.uk/) were compared to SEC data for 16 representative T. maritima proteins whose structures were solved at the JCSG, revealing an inconsistency in five cases. Herein, we report that SEC can be used to validate or disprove PQS-derived oligomeric models. A substantial amount of associated SEC and structural data should enable us to use certain PQS parameters to gauge the accuracy of these computational models and to generally improve their predictions.

Research paper thumbnail of Functional Analysis of Substrate and Cofactor Complex Structures of a Thymidylate Synthase-Complementing Protein

Research paper thumbnail of Probing the Multidomain Structure of the Type I Regulatory Subunit of cAMP-Dependent Protein Kinase Using Mutational Analysis: Role and Environment of Endogenous Tryptophans

Biochemistry, 2000

The regulatory R-subunit of cAMP-dependent protein kinase (cAPK) is a thermostable multidomain pr... more The regulatory R-subunit of cAMP-dependent protein kinase (cAPK) is a thermostable multidomain protein. It contains a dimerization domain at the N-terminus followed by an inhibitor site that binds the catalytic C-subunit and two tandem cAMP-binding domains (A and B). Two of the three tryptophans in the RIalpha subunit, Trp188 and Trp222, lie in cAMP-binding domain A while Trp260 lies at the junction between domains A and B. The unfolding of wild-type RIalpha (wt-RI), monitored by intrinsic fluorescence, was described previously [Leon, D. A., Dostmann, W. R. G., and Taylor, S. S. (1991) Biochemistry 30, 3035 (1)]. To determine the environment of each tryptophan and the role of the adjacent domain in folding and stabilization of domain A, three point mutations, W188Y, W222Y, and W260Y, were introduced. The secondary structure of wt-RI and the point mutants has been studied by far-UV circular dichroism spectropolarimetry (CD). The CD spectra of wt-RI and the three point mutants are practically identical, and the thermal unfolding behavior is very similar. Intrinsic fluorescence and iodide quenching in the presence of increasing urea established that: (a) Trp222 is the most buried, whereas Trp188 is the most exposed to solvent; (b) Trp260 accounts for the quenching of fluorescence when cAMP is bound; and (c) Trp222 contributes most to the intrinsic fluorescence of the wt-RI-subunit, while Trp188 contributes least. For wt-RI, rR(W188Y), and rR(W260Y), removal of cAMP causes a destabilization, while excess cAMP stabilizes these three proteins. In contrast, rR(W222Y) was not stabilized by excess cAMP.

Research paper thumbnail of Conformational Differences Among Solution Structures of the Type Iα, IIα and IIβ Protein Kinase A Regulatory Subunit Homodimers: Role of the Linker Regions

Journal of Molecular Biology, 2004

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are mu... more The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIa, RIIa, and RIIb homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIa and RIIb homodimers have very extended, rod-like shapes, whereas the RIa homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ia or IIa R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.

Research paper thumbnail of Amino acid variant in the kinase binding domain of dual-specific A kinase-anchoring protein 2: A disease susceptibility polymorphism

Proceedings of The National Academy of Sciences, 2003

The focus of human genetics in recent years has shifted toward identifying genes that are involve... more The focus of human genetics in recent years has shifted toward identifying genes that are involved in the development of common diseases such as cancer, diabetes, cardiovascular diseases, and Alzheimer's disease. Because many complex diseases are late-onset, the frequencies of disease susceptibility alleles are expected to decrease in the healthy elderly individuals of the population at large because of their contribution to disease morbidity and͞or mortality. To test this assumption, we compared allele frequencies of 6,500 singlenucleotide polymorphisms (SNPs) located in Ϸ5,000 genes between DNA pools of age-stratified healthy, European-American individuals.

Research paper thumbnail of Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family

Journal of Molecular Evolution, 2002

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequenc... more The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by ␤-strands 6 and 7 of the crystal structures of bovine RI␣ and rat RII␤ (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence,

Research paper thumbnail of Consequences of cAMP-Binding Site Mutations on the Structural Stability of the Type I Regulatory Subunit of cAMP-Dependent Protein Kinase

Biochemistry, 2000

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with ... more The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.

Research paper thumbnail of Domain Organization of D-AKAP2 Revealed by Enhanced Deuterium Exchange-Mass Spectrometry (DXMS

Journal of Molecular Biology, 2002

Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-... more Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfolding regarding specific binding motifs, very little is known about the overall structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Similar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto a-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site.

Research paper thumbnail of Isoform Specific Differences in Binding of a Dual-Specificity A-Kinase Anchoring Protein to Type I and Type II Regulatory Subunits of PKA

Biochemistry, 2003

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent... more Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIR than to RIR. The higher affinity for RIIR was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIR and RIIR was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIR was independent of solution ionic strength, whereas RIIR had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIR but not to RIIR. These isoform-specific differences provide a foundation for developing isoformspecific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring. † This work was funded by an NIH grant (DK-54441) to S.S.T. L.L.B. was funded by an NIH training grant (5T32 DK-07233).

Research paper thumbnail of Isoform Specific Differences in Binding of a Dual-Specificity A-Kinase Anchoring Protein to Type I and Type II Regulatory Subunits of PKA †

Biochemistry, 2003

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent... more Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIR than to RIR. The higher affinity for RIIR was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIR and RIIR was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIR was independent of solution ionic strength, whereas RIIR had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIR but not to RIIR. These isoform-specific differences provide a foundation for developing isoformspecific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring. † This work was funded by an NIH grant (DK-54441) to S.S.T. L.L.B. was funded by an NIH training grant (5T32 DK-07233).

Research paper thumbnail of Protein Biophysical Properties that Correlate with Crystallization Success in Thermotoga maritima: Maximum Clustering Strategy for Structural Genomics

Journal of Molecular Biology, 2004

Cost and time reduction are two of the driving forces in the development of new strategies for pr... more Cost and time reduction are two of the driving forces in the development of new strategies for protein crystallization and subsequent structure determination. Here, we report the analysis of the Thermotoga maritima proteome, in which we compare the proteins that were successfully expressed, purified and crystallized versus the rest of the proteome. This set of almost 500 proteins represents one of the largest, internally consistent, protein expression and crystallization datasets available. The analysis shows that individual parameters, such as isoelectric point, sequence length, average hydropathy, low complexity regions (SEG), and combinations of these biophysical properties for crystallized proteins define a distinct subset of the T. maritima proteome. The distribution profiles of the various biophysical properties in the expression/crystallization set are then used to extract rules to improve target selection and improve the efficiency and output of structural genomics, as well as general structural biology efforts.

Research paper thumbnail of The Structure of a Eukaryotic Nicotinic Acid Phosphoribosyltransferase Reveals Structural Heterogeneity among Type II PRTases

Structure, 2005

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for cellular redox reactions and... more Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for cellular redox reactions and can act as an important substrate in numerous biological processes. As a result, nature has evolved multiple biosynthetic pathways to meet this high chemical demand. In Saccharomyces cerevisiae, the NAD salvage pathway relies on the activity of nicotinic acid phosphoribosyltransferase (NAPRTase), a member of the phosphoribosyltransferase (PRTase) superfamily. Here, we report the structure of a eukaryotic (yeast) NAPRTase at 1.75 A resolution (locus name: YOR209C, gene name: NPT1). The structure reveals a two-domain fold that resembles the architecture of quinolinic acid phosphoribosyltransferases (QAPRTases), but with completely different dispositions that provide evidence for structural heterogeneity among the Type II PRTases. The identification of a third domain in NAPRTases provides a structural basis and possible mechanism for the functional modulation of this family of enzymes by ATP.

Research paper thumbnail of Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

European Biophysics Journal With Biophysics Letters, 2004

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking ... more Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N2 showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.

Research paper thumbnail of Shotgun crystallization strategy for structural genomics: An Optimized two-tiered crystallization screen against the thermotogamaritima proteome

Acta Crystallographica Section D-biological Crystallography, 2003

Research paper thumbnail of High-throughput Protein Production for X-ray Crystallography and Use of Size Exclusion Chromatography to Validate or Refute Computational Biological Unit Predictions

Journal of Structural and Functional Genomics, 2005

The production of large numbers of highly purified proteins for X-ray crystallography is a signif... more The production of large numbers of highly purified proteins for X-ray crystallography is a significant bottleneck in structural genomics. At the Joint Center for Structural Genomics (JCSG; http://www.jcsg.org), specific automated protein expression, purification, and analytical methods are being utilized to study the proteome of Thermotoga maritima. Anion exchange and size exclusion chromatography (SEC), intended for the production of highly purified proteins, have been automated and the procedures are described here in detail. Analytical SEC has been included as a standard quality control test. A biological unit (BU) is the macromolecule that has been proven or is presumed to be functional. Correct assignment of BUs from protein structures can be difficult. BU predictions obtained via the Protein Quaternary Structure file server (PQS; http://pqs.ebi.ac.uk/) were compared to SEC data for 16 representative T. maritima proteins whose structures were solved at the JCSG, revealing an inconsistency in five cases. Herein, we report that SEC can be used to validate or disprove PQS-derived oligomeric models. A substantial amount of associated SEC and structural data should enable us to use certain PQS parameters to gauge the accuracy of these computational models and to generally improve their predictions.

Research paper thumbnail of Functional Analysis of Substrate and Cofactor Complex Structures of a Thymidylate Synthase-Complementing Protein

Research paper thumbnail of Probing the Multidomain Structure of the Type I Regulatory Subunit of cAMP-Dependent Protein Kinase Using Mutational Analysis: Role and Environment of Endogenous Tryptophans

Biochemistry, 2000

The regulatory R-subunit of cAMP-dependent protein kinase (cAPK) is a thermostable multidomain pr... more The regulatory R-subunit of cAMP-dependent protein kinase (cAPK) is a thermostable multidomain protein. It contains a dimerization domain at the N-terminus followed by an inhibitor site that binds the catalytic C-subunit and two tandem cAMP-binding domains (A and B). Two of the three tryptophans in the RIalpha subunit, Trp188 and Trp222, lie in cAMP-binding domain A while Trp260 lies at the junction between domains A and B. The unfolding of wild-type RIalpha (wt-RI), monitored by intrinsic fluorescence, was described previously [Leon, D. A., Dostmann, W. R. G., and Taylor, S. S. (1991) Biochemistry 30, 3035 (1)]. To determine the environment of each tryptophan and the role of the adjacent domain in folding and stabilization of domain A, three point mutations, W188Y, W222Y, and W260Y, were introduced. The secondary structure of wt-RI and the point mutants has been studied by far-UV circular dichroism spectropolarimetry (CD). The CD spectra of wt-RI and the three point mutants are practically identical, and the thermal unfolding behavior is very similar. Intrinsic fluorescence and iodide quenching in the presence of increasing urea established that: (a) Trp222 is the most buried, whereas Trp188 is the most exposed to solvent; (b) Trp260 accounts for the quenching of fluorescence when cAMP is bound; and (c) Trp222 contributes most to the intrinsic fluorescence of the wt-RI-subunit, while Trp188 contributes least. For wt-RI, rR(W188Y), and rR(W260Y), removal of cAMP causes a destabilization, while excess cAMP stabilizes these three proteins. In contrast, rR(W222Y) was not stabilized by excess cAMP.

Research paper thumbnail of Conformational Differences Among Solution Structures of the Type Iα, IIα and IIβ Protein Kinase A Regulatory Subunit Homodimers: Role of the Linker Regions

Journal of Molecular Biology, 2004

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are mu... more The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIa, RIIa, and RIIb homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIa and RIIb homodimers have very extended, rod-like shapes, whereas the RIa homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ia or IIa R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.

Research paper thumbnail of Amino acid variant in the kinase binding domain of dual-specific A kinase-anchoring protein 2: A disease susceptibility polymorphism

Proceedings of The National Academy of Sciences, 2003

The focus of human genetics in recent years has shifted toward identifying genes that are involve... more The focus of human genetics in recent years has shifted toward identifying genes that are involved in the development of common diseases such as cancer, diabetes, cardiovascular diseases, and Alzheimer's disease. Because many complex diseases are late-onset, the frequencies of disease susceptibility alleles are expected to decrease in the healthy elderly individuals of the population at large because of their contribution to disease morbidity and͞or mortality. To test this assumption, we compared allele frequencies of 6,500 singlenucleotide polymorphisms (SNPs) located in Ϸ5,000 genes between DNA pools of age-stratified healthy, European-American individuals.

Research paper thumbnail of Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family

Journal of Molecular Evolution, 2002

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequenc... more The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by ␤-strands 6 and 7 of the crystal structures of bovine RI␣ and rat RII␤ (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence,

Research paper thumbnail of Consequences of cAMP-Binding Site Mutations on the Structural Stability of the Type I Regulatory Subunit of cAMP-Dependent Protein Kinase

Biochemistry, 2000

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with ... more The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.

Research paper thumbnail of Domain Organization of D-AKAP2 Revealed by Enhanced Deuterium Exchange-Mass Spectrometry (DXMS

Journal of Molecular Biology, 2002

Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-... more Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfolding regarding specific binding motifs, very little is known about the overall structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Similar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto a-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site.

Research paper thumbnail of Isoform Specific Differences in Binding of a Dual-Specificity A-Kinase Anchoring Protein to Type I and Type II Regulatory Subunits of PKA

Biochemistry, 2003

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent... more Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIR than to RIR. The higher affinity for RIIR was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIR and RIIR was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIR was independent of solution ionic strength, whereas RIIR had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIR but not to RIIR. These isoform-specific differences provide a foundation for developing isoformspecific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring. † This work was funded by an NIH grant (DK-54441) to S.S.T. L.L.B. was funded by an NIH training grant (5T32 DK-07233).