The majority of known proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. Here we introduce an approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques. The encapsulation of a protein in a reverse micelle dissolved in a low-viscosity fluid allows it to tumble as fast as a much smaller protein. The approach is demonstrated and validated with the protein ubiquitin encapsulated in reverse micelles prepared in a variety of alkane solvents.

The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP.ATR (ATM and Rad3-related) kinase is a protein kinase that coordinates cellular responses to genotoxic stress. ATR activation occurs primarily in S phase due to replication stress induced by DNA-damaging agents or replication inhibitors. More specifically, ATR activation is stimulated when the replication machinery encounters a DNA lesion and becomes uncoupled (the helicase continues to unwind DNA while the polymerase becomes stalled at the site of DNA damage) (9).The critical factor that promotes ATR activation is believed to be the accumulation of RPA (replication protein A)-coated single-stranded DNA (ssDNA) (11,33,43). At least two separate checkpoint complexes accumulate in distinct foci that colocalize with RPA. Rad17, a PCNA-like clamp loader protein, is recruited to RPA-ssDNA and loads the Rad9-Rad1-Hus1 checkpoint clamp at the junction of double-stranded and single-stranded DNA (4,14,53). Independently, ATR is recruited by ATR-interacting protein (ATRIP), which binds the RPA-ssDNA that accumulates at DNA lesions (3,15,37,52).ATRIP is required for ATR function, and mutation of either ATR or ATRIP causes the same phenotypes (3, 12). The strict requirement for ATRIP is conserved in Schizosaccharomyces pombe (Rad3 and Rad26), Saccharomyces cerevisiae (Mec1 and Ddc2/Lcd1/Pie1), and Xenopus laevis (xATR and xATRIP) (13,38,41,51). An N-terminal domain of ATRIP binds RPAssDNA and is necessary for stable ATR-ATRIP localization to damage-induced nuclear foci (3, 25).The ATR signaling pathway is currently viewed as an important target for the development of cancer therapies (10,22,24,32,34). However, the mechanism by which ATR is activated remains unclear. Localization to sites of DNA damage or replication stress has been suggested to be essential and perhaps sufficient to promote ATR signaling. However, mutations in ATRIP that disrupt the stable RPA-A...

Modular proteins with multiple domains tethered by flexible linkers have variable global archiectures. Using the eukaryotic ssDNA binding protein, Replication Protein A (RPA), we demonstrate that NMR spectroscopy is a powerful tool to characterize the remodeling of architecture in different functional states. The first direct evidence is obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain that may help explain its damage-dependent phosphorylation.The progression of DNA replication and repair requires the coordinated action of dynamic, multi-protein assemblies. We have previously proposed a critical role for proteins composed of multiple, flexibly attached domains in facilitating the action of these dynamic complexes 1 . Because these proteins can undergo intra-and inter-domain rearrangements, they are able to interact optimally with the ever-changing substrate landscape present during DNA processing. RPA is a prototypical modular multi-domain DNA processing protein with flexible linkers of various lengths (Figure 1). The trimer core is a compact assembly of three OB-fold domains (RPA70C/32D/14) to which is appended the disordered RPA32N functional domain, the RPA32C winged-helix domain, and the tandem RPA70AB and the RPA70N OB-fold NMR spectroscopy in solution is a powerful tool for characterizing proteins under conditions that preserve intrinsic dynamic properties. The advent of TROSY, CRINEPT and related experimental approaches 3 has vastly increased the upper limit of molecular masses accessible to study by NMR. Examples range from the globular malate synthase (82 kDa) to the oligomeric GroEL-GroES complex (872 kDa) to highly flexible domains from the ribosome (>2.5 MDa) 4 . In the case of RPA (116 kDa) and many other multi-domain proteins, modularity and interdomain flexibility are the critical properties that enable characterization of dynamic architectures by NMR.To illustrate the analytical framework, results are presented first for RPA70NAB (M r 45.8 kDa), which has an asymmetric arrangement with a 70-residue N-A linker and a 10-residue A-B linker (Figure 1). The 15 N-1 H TROSY-HSQC spectrum of 15 N-enriched RPA70NAB reveals the presence of over 370 of the 400 expected signals from 422 residues (Figure 2). The signals from each of the three domains appear in positions remarkably similar to those in NMR spectra of the three isolated domains ( Figure S1). Thus, all three domains are structurally independent and resonance assignments can be transferred from the isolated domains to RPA70NAB 5 . NMR is highly sensitive to differences in the degree of inter-domain flexibility; the signals from the A and B domains are substantially weaker than the signals from the N domain, even though all three domains are approximately the same mass ( Figure 2). The differences arise from the fact that although the A and B domains are structurally independent, the short A-B tether partially restricts their motions, whereas the much longer N-A tether e...

No abstract

Calmodulin binds to amphiphilic, helical peptides of a variety of amino-acid sequences. These peptides are usually positively charged, although there is spectroscopic evidence that at least one neutral peptide binds. The complex between calmodulin and one of its natural target peptides, the binding site for calmodulin on smooth muscle myosin light-chain kinase (RS20), has been investigated by crystallography and NMR which have characterized the interactions between the ligand and the protein. From these data, it appears that the calmodulin-binding surface is sterically malleable and van der Waals forces probably dominate the binding. To explore further this apparently permissive binding, we investigated the chiral selectivity of calmodulin using synthesized analogues of melittin and RS20 that consisted of only D-amino acids. Fluorescence and NMR measurements show that D-melittin and D-RS20 both bind avidly to calmodulin, probably in the same general binding site as that for peptides having all L-amino acids. The calmodulin-peptide binding surface is therefore remarkably tolerant sterically. Our results suggest a potentially useful approach to the design of non-hydrolysable or slowly hydrolysable intracellular inhibitors of calmodulin.

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE 01-03-2008 REPORT TYPE Annual Summary DATES COVERED PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBERVanderbilt University Medical Center Nashville, TN 37232 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTESOriginal contains colored plates: ALL DTIC reproductions will be in black and white. ABSTRACTGenomic instability is a hallmark of breast cancer cells. DNA damage checkpoints are critical for the prevention of genomic instability and breast cancer. The ATR checkpoint kinase is activated in response to exogenous and endogenous DNA damage, and phosphorylates downsteam substrates such as BRCA1 and p53 to promote cell cycle arrest DNA repair, and apoptosis. ATR exists in a complex with ATR-interacting protein (ATRIP). In response to DNA damage, the ATR-ATRIP complex is recruited to DNA lesions in part through an interaction between ATRIP and the single-stranded DNA binding protein RPA (Replication Protein A). We report the identification of a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs by biochemical mapping of the RPA binding site in combination with NMR, mutagenesis, and computational modeling studies. Mutations in the CRD of the yeast ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA damaging agents, and partially compromise checkpoint signaling. We have also defined a conserved TopBP1 interacting region in ATRIP that is necessary for ATR activation. Finally, we have discovered a PIKK regulatory domain (PRD) in ATR that is required for its activation by TopBP1 and cellular viability. Thus, our results support a multi-step model for ATR activation that requires separable localization and activation functions of ATRIP, and helps to explain how cells maintain genome stability and prevent tumorigenesis....

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBERVanderbilt University Medical Center Nashville, TN 37232 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTESOriginal contains colored plates: ALL DTIC reproductions will be in black and white. ABSTRACTThe ATR (ATM and Rad3-Related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (Replication Protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs has been identified by biochemical mapping of the RPA binding site in combination with NMR, mutagenesis and computational modeling. Mutations in the CRD of the yeast ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of TopBP1 to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support a multi-step model for ATR activation that requires separable localization and activation functions of ATRIP.