Disordered Loop Region Research Articles

Identification of a Cryptic Pocket in Methionine Aminopeptidase-II Using Adaptive Bandit Molecular Dynamics Simulations and Markov State Models.

Methionine aminopeptidase-II (MetAP-II) is a metalloprotease, primarily responsible for the cotranslational removal of the N-terminal initiator methionine from the nascent polypeptide chain during protein synthesis. MetAP-II has been implicated in angiogenesis and endothelial cell proliferation and is therefore considered a validated target for cancer therapeutics. However, there is no effective drug available against MetAP-II. In this study, we employ Adaptive Bandit molecular dynamics simulations to investigate the structural dynamics of the apo and ligand-bound MetAP-II. Our results focus on the dynamic behavior of the disordered loop that is not resolved in most of the crystal structures. Further analysis of the conformational flexibility of the disordered loop reveals a hidden cryptic pocket that is predicted to be potentially druggable. The network analysis indicates that the disordered loop region has a direct signaling route to the active site. These findings highlight a new way to target MetAP-II by designing inhibitors for the allosteric site within this disordered loop region.

Biophysical insights into the dimer formation of human Sirtuin 2

AbstractSirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild‐type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 (hSIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT hSIRT2 HDAC (residues 52–357) forms a homodimer in a concentration‐dependent manner with a dimer–monomer dissociation constant of 8.3 ± 0.5 μM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of hSIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of hSIRT2 HDAC revealed that some non‐dimerizing mutants exhibited deacetylation activity for the N‐terminal peptide of histone H3, similar to the wild type. The hSIRT2 HDAC mutant Δ292–306, which lacks a SIRT2‐specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X‐ray structure of the mutant Δ292–306 was almost identical to that of the WT hSIRT2 HDAC bound to an inhibitor. These results indicate that hSIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of hSIRT2 based on our biophysical experimental results.

Substrate-induced condensation activates plant TIR domain proteins

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR–NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

A dynamic template complex mediates Munc18-chaperoned SNARE assembly.

Munc18 chaperones assembly of three membrane-anchored soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a four-helix bundle to mediate membrane fusion between vesicles and plasma membranes, leading to neurotransmitter or insulin release, GLUT4 translocation, or other exocytotic processes. Yet, the molecular mechanism underlying chaperoned SNARE assembly is not well understood. Recent evidence suggest that Munc18-1 and Munc18-3 simultaneously bind their cognate SNAREs to form ternary template complexes - Munc18-1:Syntaxin-1:VAMP2 for synaptic vesicle fusion and Munc18-3:Syntaxin-4:VAMP2 for GLUT4 translocation and insulin release, which facilitate binding of SNAP-25 or SNAP-23 to conclude SNARE assembly. Here, we further investigate the structure, dynamics, and function of the template complexes using optical tweezers. Our results suggest that the synaptic template complex transitions to an activated state with a rate of ∼0.05 s-1 and ∼6.8 kBT higher energy for efficient SNAP-25 binding. The transition depends upon the linker region of syntaxin-1 upstream of its helical bundle-forming SNARE motif. In addition, the template complex is stabilized by a poorly characterized disordered loop region in Munc18-1. While the synaptic template complex efficiently binds both SNAP-25 and SNAP-23, the GLUT4 template complex strongly favors SNAP-23 over SNAP-25, despite similar stabilities of their assembled SNARE bundles. Together, our data demonstrate that a highly dynamic template complex mediates efficient and specific SNARE assembly.

Kinetochore-microtubule attachment in human cells is regulated by the interaction of a conserved motif of Ska1 with EB1

The kinetochore establishes the linkage between chromosomes and the spindle microtubule plus ends during mitosis. In vertebrates, the spindle-kinetochore-associated (Ska1,2,3) complex stabilizes kinetochore attachment with the microtubule plus ends, but how Ska is recruited to and stabilized at the kinetochore-microtubule interface is not understood. Here, our results show that interaction of Ska1 with the general microtubule plus end-associated protein EB1 through a conserved motif regulates Ska recruitment to kinetochores in human cells. Ska1 forms a stable complex with EB1 via interaction with the motif in its N-terminal disordered loop region. Disruption of this interaction either by deleting or mutating the motif disrupts Ska complex recruitment to kinetochores and induces chromosome alignment defects, but it does not affect Ska complex assembly. Atomic-force microscopy imaging revealed that Ska1 is anchored to the C-terminal region of the EB1 dimer through its loop and thereby promotes formation of extended structures. Furthermore, our NMR data showed that the Ska1 motif binds to the residues in EB1 that are the binding sites of other plus end targeting proteins that are recruited to microtubules by EB1 through a similar conserved motif. Collectively, our results demonstrate that EB1-mediated Ska1 recruitment onto the microtubule serves as a general mechanism for the formation of vertebrate kinetochore-microtubule attachments and metaphase chromosome alignment.

A dynamic template complex mediates Munc18-chaperoned SNARE assembly

Munc18 chaperones assembly of three membrane-anchored soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a four-helix bundle to mediate membrane fusion between vesicles and plasma membranes, leading to neurotransmitter or insulin release, glucose transporter (GLUT4) translocation, or other exocytotic processes. Yet, the molecular mechanism underlying chaperoned SNARE assembly is not well understood. Recent evidence suggests that Munc18-1 and Munc18-3 simultaneously bind their cognate SNAREs to form ternary template complexes - Munc18-1:Syntaxin-1:VAMP2 for synaptic vesicle fusion and Munc18-3:Syntaxin-4:VAMP2 for GLUT4 translocation and insulin release, which facilitate the binding of SNAP-25 or SNAP-23 to conclude SNARE assembly. Here, we further investigate the structure, dynamics, and function of the template complexes using optical tweezers. Our results suggest that the synaptic template complex transitions to an activated state with a rate of 0.054 s-1 for efficient SNAP-25 binding. The transition depends upon the linker region of syntaxin-1 upstream of its helical bundle-forming SNARE motif. In addition, the template complex is stabilized by a poorly characterized disordered loop region in Munc18-1. While the synaptic template complex efficiently binds both SNAP-25 and SNAP-23, the GLUT4 template complex strongly favors SNAP-23 over SNAP-25, despite the similar stabilities of their assembled SNARE bundles. Together, our data demonstrate that a highly dynamic template complex mediates efficient and specific SNARE assembly.

Autoregulation of GPCR conformation and selective G protein activation by an intrinsically disordered loop region

While the structural mechanism of G protein-coupled receptor (GPCR) activation is well studied, there is sparse information concerning the functional role of the intrinsically disordered intracellular loops of the receptor in said mechanism. We show that the third intracellular loop (ICL3) of the beta 2 adrenergic receptor (β2AR) autoregulates receptor activation and G protein-mediated signaling. Using a combination of time resolved fluorescence spectroscopy and molecular dynamics simulations, we find that ICL3 is in an equilibrium between an ensemble of closed and open conformational states.

Secondary Mutation-Induced Alternative Splicing Suppresses RNA Splicing Defect of the jhs1 Mutant.

jing he sheng1 (jhs1) is a mutant of the DNA2 homolog in Arabidopsis (Arabidopsis thaliana), which was previously identified as being involved in DNA damage repair, cell cycle regulation, and meristem maintenance. A mutation at the 3' intron splice site of the 11th intron causes alternative splicing of this intron at two other sites, which results in frame shifts and premature stop codons. Here, we screened suppressors of jhs1 to further study the function and regulatory networks of JHS1 Three suppressors with wild-type-like phenotypes were obtained. Sequencing analysis results showed that each of the suppressors has a second mutation in jhs1 that causes further alternative splicing of the intron and corrects the shifted reading frame with small insertions. Precursor mRNA sequence analysis and intron splice site evaluation results suggested that intron splicing was disturbed in the suppressors, and this switched the splice site, resulting in small insertions in the coding regions of JHS1. Structural analysis of JHS1 suggested that the insertions are in a disordered loop region of the DNA2 domain and do not seem to have much deleterious effect on the function of the protein. This work not only has implications for the evolution of protein sequences at exon junctions but also provides a strategy to study the mechanism of precursor mRNA splicing.

Concomitant disorder and high-affinity zinc binding in the human zinc- and iron-regulated transport protein 4 intracellular loop.

The human zinc- and iron-regulated transport protein 4 (hZIP4) protein is the major plasma membrane protein responsible for the uptake of zinc in the body, and as such it plays a key role in cellular zinc homeostasis. hZIP4 plasma membrane levels are regulated through post-translational modification of its large, disordered, histidine-rich cytosolic loop (ICL2) in response to intracellular zinc concentrations. Here, structural characteristics of the isolated disordered loop region, both in the absence and presence of zinc, were investigated using nuclear magnetic resonance (NMR) spectroscopy. NMR chemical shifts, coupling constants and temperature coefficients of the apoprotein, are consistent with a random coil with minor propensities for transient polyproline Type II helices and β-strand in regions implicated in post-translational modifications. The ICL2 protein remains disordered upon zinc binding, which induces exchange broadening. Paramagnetic relaxation enhancement experiments reveal that the histidine-rich region in the apoprotein makes transient tertiary contacts with predicted post-translational modification sites. The residue-specific data presented here strengthen the relationship between hZIP4 post-translational modifications, which impact its role in cellular zinc homeostasis, and zinc sensing by the intracellular loop. Furthermore, the zinc sensing mechanism employed by the ICL2 protein demonstrates that high-affinity interactions can occur in the presence of conformational disorder.

NMR assignments for the insertion domain of bacteriophage Sf6 coat protein.

The P22 bacteriophage group is a subgroup of the λ phagesupercluster, comprised of the three major sequence types Sf6, P22, and CUS-3, based on their capsid proteins. Our goal is to investigate the extent to which structure-function relationships are conserved for the viral coat proteins and I-domains in this subgroup. Sf6 is a phage that infects the human pathogen Shigella flexneri. The coat protein of Sf6 assembles into a procapsid, which further undergoes maturation during DNA packaging into an infectious virion. The Sf6 coat protein contains a genetically inserted domain, termed the I-domain, similar to the ones present in the P22 and CUS-3 coat proteins. Based on the P22 example, I-domains play important functional roles in capsid assembly, stability, viability, and size-determination. Here we report the 1H, 15N, and 13C chemical shift assignments for the I-domain of the Sf6 phage coat protein. Chemical shift-based secondary structure prediction and hydrogen-bond patterns from a long-range HNCO experiment indicate that the Sf6 I-domain adopts a 6-stranded β-barrel fold like those of P22 and CUS-3 but with important differences, including the absence of the D-loop that is critical for capsid assembly and the addition of a novel disordered loop region.

Relationship between Structural Flexibility and Function in the C-Terminal Region of the Heparin-Binding Domain of VEGF165

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Previously, we reported that triamterene (Trm) inhibits VEGF-amyloid β (Aβ) interactions without affecting other biological activities of VEGF or Aβ [Jeong, K.-W., et al. (2011) Biochemistry 50, 4843-4854]. We further showed that molecular motions in the N-terminal disordered loop region of the heparin-binding domain (HBD) are important for interaction with Trm. To investigate the importance of motion at the C-terminal domain of HBD, we constructed a binding model of HBD with heparin octasaccharide (HOS) based on measurements of chemical shift changes and docking studies. Furthermore, the dynamic properties of the HBD-HOS and HBD-Trm-HOS complexes were assessed by measuring spin relaxation rates. The results showed that the HOS-binding site is composed of two basic clusters consisting of side chains of residues R13, R14, and K15 and residues K30, R35, and R49. When HOS binds, values for the heteronuclear nuclear Overhauser effect near HOS-binding sites increased dramatically. CPMG (Carr-Purcell-Meiboom-Gill sequence) experiments as well as an R2 relaxation experiment were undertaken to understand millisecond time-scale motions in HBD. There is large relaxation dispersion of residues at Trm- and HOS-binding sites in free HBD. C-Terminal residues such as S34, C48, and D51 near the HOS-binding sites continued to exhibit slow conformational motions in the HBD-Trm complex, while those slow motions disappeared in the bound conformation of HBD with HOS. Collectively, our results demonstrate that the inherent structural flexibilities of the C-terminal region of the HBD are important in the heparin binding process and that Trm does not inhibit VEGF-heparin interactions necessary for the biological activities of VEGF.

Dynamics of a Heparin-Binding Domain of VEGF165 Complexed with Its Inhibitor Triamterene

Vascular endothelial growth factor (VEGF), which has neurotrophic and neuroprotective effects in addition to its major role in angiogenesis, interacts with Aβ and accumulates in the senile plaques of Alzheimer's disease (AD) patients' brains. It is known that Aβ binds to the heparin-binding domain (HBD) of the 165-amino acid VEGF variant, VEGF(165). In this study, we showed that triamterene (Trm) inhibits VEGF--Aβ interaction without affecting other biological activities of VEGF or Aβ. We investigated the importance of structural and dynamic features of HBD for its molecular-recognition processes. The binding model of HBD and Trm was constructed based on measurements of chemical shift changes and docking study. The results showed that the loop region (S11-L17) and F18 at the beginning of the first β-sheet in the HBD constitute the inhibitor binding site. The N1 atom of pteridine ring of Trm forms hydrogen bonding with backbone amide proton of R13, and the phenyl ring took part in a hydrophobic interaction with the aromatic ring of F18. To investigate the functional importance of the inherent structural flexibility of the HBD in VEGF, the dynamic properties of free HBD and HBD--Trm complex were assessed by measuring spin relaxation rates, and the backbone dynamics were investigated by model-free analysis. The residues in the disordered loop region of the N-terminus exhibited conformational exchanges in free HBD, and flexibility of this loop region decreased dramatically upon binding to Trm, suggesting that Aβ as well as inhibitor may recognize these unique dynamic features of the HBD. Furthermore, C-terminal residues continued to exhibit slow conformational motions, even in the HBD--Trm complex, implying that these motions at the C-terminus of the HBD might be important for interactions with heparin molecules. The flexibility of HBD demonstrated here should be essential for VEGF function and interaction with other protein partners.

A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity

Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P2(1) with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6 A resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P2(1) crystal form uncovered major conformational changes (i) in beta-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the beta-barrel and (iii) in the extended C-terminal segment, which is attached to the beta-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family.

The Major Human Pregnane X Receptor (PXR) Splice Variant, PXR.2, Exhibits Significantly Diminished Ligand-Activated Transcriptional Regulation

The pregnane X receptor (PXR; PXR.1) can be activated by structurally diverse lipophilic ligands. PXR.2, an alternatively spliced form of PXR, lacks 111 nucleotides encoding 37 amino acids in the ligand binding domain. PXR.2 bound a classic CYP3A4 PXR response element (PXRE) in electrophoretic mobility shift assays, but transfected PXR.2 failed to transactivate a CYP3A4-promoter-luciferase reporter plasmid in HepG2 cells treated with various PXR ligands. Cotransfection experiments showed that PXR.2 behaved as a dominant negative, interfering with PXR.1/rifampin activation of CYP3A4-PXRE-LUC. In HepG2 and LS180 cells stably transduced with PXR.1, PXR target genes (CYP3A4, MDR1, CYP2B6, and UGT1A1) were higher than mock-transduced cells in the absence of ligand and were further induced in the presence of rifampin. In contrast, PXR.2 stably introduced into the same host cells failed to induce target genes over levels in mock-transfected cells after drug treatment. Our homology modeling suggests that ligands bind PXR.1 more favorably, probably because of the presence of a key disordered loop region, which is missing in PXR.2. Yeast two-hybrid assays revealed that, even in the presence of ligand, the corepressors remain tightly bound to PXR.2, and coactivators are unable to bind at helix 12. In summary, PXR.2 can bind to PXREs but fails to transactivate target genes because ligands do not bind the ligand binding domain of PXR.2 productively, corepressors remain tightly bound, and coactivators are not recruited to PXR.2.

Crystal Structure of the VP4 Protease from Infectious Pancreatic Necrosis Virus Reveals the Acyl-Enzyme Complex for an Intermolecular Self-cleavage Reaction

Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH(2)-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-A resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser(633)Ogamma forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala(716), of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-A resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity ( approximately 19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.

Crystallographic Study of the Recombinant Flavin-Binding Domain of Baker's Yeast Flavocytochrome b2: Comparison with the Intact Wild-Type Enzyme

Flavocytochrome b(2) catalyzes the oxidation of L-lactate to pyruvate and the transfer of electrons to cytochrome c. The enzyme consists of a flavin-binding domain, which includes the active site for lacate oxidation, and a b(2)-cytochrome domain, required for efficient cytochrome c reduction. To better understand the structure and function of intra- and interprotein electron transfer, we have determined the crystal structure of the independently expressed flavin-binding domain of flavocytochrome b(2) to 2.50 A resolution and compared this with the structure of the intact enzyme, redetermined at 2.30 A resolution, both structures being from crystals cooled to 100 K. Whereas there is little overall difference between these structures, we do observe significant local changes near the interface region, some of which impact on amino acid side chains, such as Arg289, that have been shown previously to have an important role in catalysis. The disordered loop region found in flavocytochrome b(2) and its close homologues remain unresolved in frozen crystals of the flavin-binding domain, implying that the presence of the b(2)-cytochrome domain is not responsible for this positional disorder. The flavin-binding domain interacts poorly with cytochrome c, but we have introduced acidic residues in the interdomain interface region with the aim of enhancing cytochrome c binding. While the mutations L199E and K201E within the flavin-binding domain resulted in unimpaired lactate dehydrogenase activity, they failed to enhance electron-transfer rates with cytochrome c. This is most likely due to the disordered loop region obscuring all or part of the surface having the potential for productive interaction with cytochrome c.

Functional roles of conserved residues in the unstructured loop of Vibrio harveyi bacterial luciferase.

Residues 257-291 of the Vibrio harveyi bacterial luciferase alpha subunit comprise a highly conserved, protease-labile, disordered loop region, most of which is unresolved in the previously determined X-ray structures of the native enzyme. This loop region has been shown to display a time- dependent proteolysis resistance upon single catalytic turnover and was postulated to undergo conformational changes during catalysis ([AbouKhair, N. K., Ziegler, M. M., and Baldwin, T. O. (1985) Biochemistry 24, 3942-3947]. To investigate the role of this region in catalysis, we have performed site-specific mutations of different conserved loop residues. In comparison with V(max) and V(max)/K(m,flavin) of the native luciferase, the bioluminescence activities of alphaG284P were decreased to 1-2% whereas those of alphaG275P and alphaF261D were reduced by 4-6 orders of magnitude. Stopped-flow results indicate that both alphaG275P and alphaF261D were able to form the 4a-hydroperoxy-FMN intermediate II but at lower yields. Both mutants also had enhanced rates for the intermediate II nonproductive dark decay and significantly compromised abilities to oxidize the decanal substrate. Additional mutations were introduced into the alphaG275 and alphaF261 positions, and the activities of the resulting mutants were characterized. Results indicate that the torsional flexibility of the alphaG275 residue and the bulky and hydrophobic nature of the alphaF261 residue were critical to the luciferase activity. Our results also support a functional role for the alpha subunit unstructured loop itself, possibly by serving as a mobile gating mechanism in shielding critical intermediates (including the excited flavin emitter) from exposure to medium.

Crystal structure of ribosomal protein L4 shows RNA-binding sites for ribosome incorporation and feedback control of the S10 operon.

Ribosomal protein L4 resides near the peptidyl transferase center of the bacterial ribosome and may, together with rRNA and proteins L2 and L3, actively participate in the catalysis of peptide bond formation. Escherichia coli L4 is also an autogenous feedback regulator of transcription and translation of the 11 gene S10 operon. The crystal structure of L4 from Thermotoga maritima at 1.7 A resolution shows the protein with an alternating alpha/beta fold and a large disordered loop region. Two separate binding sites for RNA are discernible. The N-terminal site, responsible for binding to rRNA, consists of the disordered loop with flanking alpha-helices. The C-terminal site, a prime candidate for the interaction with the leader sequence of the S10 mRNA, involves two non-consecutive alpha-helices. The structure also suggests a C-terminal protein-binding interface, through which L4 could be interacting with protein components of the transcriptional and/or translational machineries.

Differential multiple-quantum relaxation arising from cross-correlated time-modulation of isotropic chemical shifts.

In this paper it is demonstrated that cross-correlated time modulation of isotropic chemical shifts ('conformational exchange') leads to differential relaxation of double- and zero-quantum coherences, respectively. Quantitative information can be obtained from the time dependence of the interconversion between the two two-spin coherences 2IxSx and 2IySy, induced by the differential relaxation. The effect is illustrated with an application to 13C,15N-labeled quail CRP2(LIM2), by studying 15N-1H(N) multiple-quantum relaxation. Significant cross-correlated fluctuations of isotropic chemical shifts were observed for residues which are part of a disordered loop region connecting two beta-strands in CRP2(LIM2). Differential 1H(N) and 15N exchange contributions to multiple-quantum relaxation observed at these sites illustrate the complex interplay between hydrogen bonding events and conformational reorientations in proteins.

Refolding, Purification, and Characterization of a Loop Deletion Mutant of Human Bcl-2 from Bacterial Inclusion Bodies

This report describes the cloning of recombinant human Bcl-2, in which the putative disordered loop region has been replaced with a flexible linker and the hydrophobic C-terminus has been replaced with a 6xHis tag (Bcl-2(6–32)-AAAA-Bcl-2(86–206)-HHHHHH, abbreviationrhBcl-2; amino acid numbering excludes the initiating methionine). This protein was expressed inEscherichia coliwhere it accumulated in insoluble form in inclusion bodies. After lysis the washed inclusion bodies were solubilized and anl-arginine assisted protein refolding route was employed to obtain biologically active protein.rhBcl-2 was purified further by nickel chelate chromatography to give protein of >95% purity, with an overall yield of 5 mg per g ofE. colicell paste. Edman sequencing showed that ∼90% of therhBcl-2 retained the initiating methionine residue. Analytical size exclusion chromatography suggested that the refolded and purifiedrhBcl-2 was monomeric in nondenaturing solution. Purified protein had an affinity for a Bax BH3 domain peptide comparable to that forin vivofolded recombinant human Bcl-2 and suppressed caspase activation in a cell-free assay for apoptosis.1H NMR spectroscopy ofrhBcl-2, both free and complexed with the Bax BH3 domain peptide, provided further evidence for the structural and functional integrity of the refolded protein. These findings parallel and extend those of Muchmoreet al.,who found that a loop deletion mutant of human Bcl-XLretained anti-apoptotic function.