EMSL: Nancy Isern's Publications

Olson A, OM Hyyti, GA Cohen, XH Ning, M Sadilek, NG Isern, and MA Portman. 2008. "Superior Cardiac Function Via Anaplerotic Pyruvate in the Immature Swine Heart After Cardiopulmonary Bypass and Reperfusion." American Journal of Physiology. Heart and Circulatory Physiology 295(6):H2315-H2320. doi:10.1152/ajpheart.00739.2008 Abstract Pyruvate produces inotropic responses in the adult reperfused heart. Pyruvate oxidation and anaplerotic entry into the citric acid cycle (CAC) via carboxylation are linked to stimulation of contractile function. The goals of this study were to determine if these metabolic pathways operate and are maintained in the developing myocardium after reperfusion. Immature male swine (age 10-18 days) were subjected to cardiopulmonary bypass (CPB). Intracoronary infusion of [2]-13C-pyruvate (to achieve a final concentration of 8 mM) was given for 35 minutes starting either during weaning (Group I), after discontinuation (Group II) or without (Control) CPB. Hemodynamic data was collected. 13C NMR spectroscopy was used to determine the fraction of pyruvate entering the CAC via pyruvate carboxylation (PC) to total CAC entry (PC plus decarboxlyation via pyruvate dehydrogenase). Liquid chromatography-mass spectrometry was used to determine total glutamate enrichment.

Dantas G, C Corrent, SL Reichow, JJ Havranek, Z Eletr, NG Isern, B Kuhlman, G Varani, E Merritt, and D Baker. 2007. "High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design." Journal of Molecular Biology 366(4):1209-1221. Abstract Recent efforts to redesign or de novo design the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol, RosettaDesign, to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal / mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we design and characterize a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations – one mutant gains over 5 kcal/mol of stability over the wild-type protein with only four amino-acid changes. We explore the relationship between force-field resolution and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using force fields of different resolution and both fixed and flexible backbone sampling protocols.

Smirnov S, NG Isern, ZG Jiang, DW Hoyt, and CJ Mcknight. 2007. "The Isolated Sixth Gelsolin Repeat and Headpiece Domain of Villin Bundle F-Actin in the Presence of Calcium and Are Linked by a 40-Residue Unstructured Sequence ." Biochemistry 46(25):7488-7496. doi:10.1021/bi700110v Abstract Villin is an F-actin regulating, modular protein with a gelsolin-like core and a distinct C-terminal 'headpiece’ domain. Localized in the microvilli of the absorptive epithelium, villin can bundle F-actin and, at higher calcium concentration, is capable of a gelsolin-like F-actin severing. The headpiece domain can, in isolation, bind F-actin and is crucial for F-actin bundling by villin. While the three-dimensional structure of the isolated headpiece is known, its conformation in the context of attachment to the villin core remains unexplored. Furthermore, the dynamics of the linkage of headpiece to the core has not been determined. To address these issues, we employ a 208 residue modular fragment of villin, D6-HP, which consists of the sixth gelsolin-like domain of villin (D6) and the headpiece (HP). We demonstrate that this protein fragment requires calcium for structural stability and, surprisingly, is capable of Ca2+-dependent F-actin bundling, suggesting that D6 contains a cryptic F-actin binding site. NMR resonance assignments and 15N-relaxation measurements of D6-HP in 5 mM Ca2+ demonstrate that D6-HP consists of two independent structural domains (D6 and HP) connected by an unfolded 40-residue linker sequence. The headpiece domain in D6-HP retains its structure and interacts with D6 domain only through the linker sequence without engaging in other interactions. Chemical shift values indicate essentially the same secondary structure elements for the D6 domain in D6-HP as in the highly homologous gelsolin domain 6. Thus, the headpiece domain of villin is structurally and functionally independent from the core domain.

Dantas G, AL Watters, B Lunde, Z Eletr, NG Isern, T Roseman, J Lipfert, S Doniach, M Tompa, B Kuhlman, BL Stoddard, G Varani, and D Baker. 2006. "Mis-translation of a Computationally Designed Protein Yields an Exceptionally Stable Homodimer: Implications for Protein Engineering and Evolution." Journal of Molecular Biology 362(5):1004-1024. doi:10.1016/j.jmb.2006.07.092 Abstract We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mistranslated and accumulates at high levels in E. coli. We used circular dichroism spectroscopy, size-exclusion chromatography, small-angle x-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting CFr protein adopts a compact, extremely-stable, obligate, symmetric, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery, and the rarity of correspondingly stable fragments in natural proteins implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mistranslated CFr sub-units to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of stable protein sub-structures.

Zhang L, M DeRider, MA McCornack, C Jao, NG Isern, T Ness, R Moyer, and PJ Liwang. 2006. "Solution structure of the complex between poxvirus-encoded CC chemokine inhibitor vCCI and human MIP-1β." Proceedings of the National Academy of Sciences of the United States of America 103(38):13985-13990. doi:10.1073/pnas.0602142103 Abstract Chemokines (chemotactic cytokines) comprise a large family of proteins that recruit and activate leukocytes, giving chemokines a major role in both the immune response and inflammation-related diseases. The poxvirus-encoded viral CC chemokine inhibitor (vCCI) binds to many CC chemokines with high affinity, acting as a potent inhibitor of chemokine action. We have used heteronuclear multidimensional NMR to determine the first structure of an orthopoxvirus vCCI in complex with a human CC chemokine MIP-1β. vCCI binds to the chemokine with 1:1 stoichiometry, using residues from its β-sheet II to interact with the a surface of MIP-1β that includes the N-terminus, the following residues in the so-called N-loop20’s region, and the 40’s loop. This structure reveals a general strategy of vCCI for selective chemokine binding, as vCCI appears to interact most stronglyinteracts most directly with residues that are conserved among a subset of CC chemokines, but are not conservednot among the other chemokine subfamilies. This structure reveals a general strategy of vCCI for selective chemokine binding. Chemokines play critical roles in the immune system, causing chemotaxis of a variety of cells to sites of infection and inflammation, as well as mediating cell homing and immune system development 1(Baggiolini 2001). To date, about 50 chemokines have been identified, and these small proteins (7-14 kDa) are believed to function by binding with endothelial or matrix glycosaminoglycans to form a concentration gradient that is then sensed by high affinity, 7-transmembrane domain G-protein coupled chemokine receptors on the surface of immune cells surface. The chemokine system is critical for host defense in healthy individuals, butand can also lead to diseases including asthma, arthritis, and atherosclerosis in the case of malfunction, often due to inappropriate inflammation and subsequent tissue damage 2(Gerard and Rollins 2001). There are four subfamilies of chemokines, CC, CXC, C, and CX3C, named for the position of conserved N-terminal cysteine residues. Members of the same subfamily often have overlapping receptor binding and cell activation ability while different subfamilies tend to functionwork on different cell subsets1{Baggiolini, 2001 #472} (REF). For example, CC chemokines mostly interact with monocytes, macrophages, T cells and eosinophils, while CXC chemokines mainly interact with neutrophils. Structures of chemokines from different subfamilies have been solved by NMR and X-ray crystallography (XXX Include Fernandez and Lolis review) 3-9{Clore, 1990 #91;Skelton, 1995 #97;Handel, 1996 #93;Crump, 1997 #92;Crump, 1998 #248;Meunier, 1997 #96;Fernandez, 2002 #496}(Clore 1990; Skelton 1995; Handel and Domaille 1996; Crump, Gong et al. 1997; Meunier 1997; Crump, Rajarathnam et al. 1998)(Clore 1990; Skelton 1995; Handel and Domaille 1996; Crump et al. 1997; Meunier 1997; Crump et al. 1998).

Kim S, Z Zhang, S Upchurch, NG Isern, and Y Chen. 2004. "Structure and DNA-Binding Sites of the SWI1 AT-rich Interaction Domain (ARID) Suggest Determinants for Sequence-Specific DNA Recognition." Journal of Biological Chemistry 279(16):16670-16676. Abstract 2 ARID is a homologous family of DNA-binding domains that occur in DNA binding proteins from a wide variety of species, ranging from yeast to nematodes, insects, mammals and plants. SWI1, a member of the SWI/SNF protein complex that is involved in chromatin remodeling during transcription, contains the ARID motif. The ARID domain of human SWI1 (also known as p270) does not select for a specific DNAsequence from a random sequence pool. The lack of sequence specificity shown by the SWI1 ARID domain stands in contrast to the other characterized ARID domains, which recognize specific AT-rich sequences. We have solved the three-dimensional structure of human SWI1 ARID using solution NMR methods. In addition, we have characterized non-specific DNA-binding by the SWI1 ARID domain. Results from this study indicate that a flexible long internal loop in ARID motif is likely to be important for sequencespecific DNA-recognition. The structure of human SWI1 ARID domain also represents a distinct structural subfamily. Studies of ARID indicate that boundary of the DNA binding structural and functional domains can extend beyond the sequence homologous region in a homologous family of proteins. Structural studies of homologous domains such as ARID family of DNA-binding domains should provide information to better predict the boundary of structural and functional domains in structural genomic studies. Key Words: ARID, SWI1, NMR, structural genomics, protein-DNA interaction.

McAteer K, A Aceves Gaona, R Michalczyk, GW Buchko, NG Isern, LA Silks, JH Miller, and MA Kennedy. 2004. "Compensating Bends in a 16 base-pair DNA Oligomer Containing a T3A3 Segment: A NMR Study of Global DNA Curvature." Biopolymers 75(6):497-511. Abstract DNA curvature but no single model has been able to explain all the experimental data. An intriguing observation is that AnTn segments ligated in phase exhibit retarded migration in polyacrylamide gel electrophoresis (PAGE) but TnAn segments do not. We have determined a high-resolution structure of a 16 base-pair DNA oligomer [d(CGAGGTTTAAACCTCG)2] containing a T3A3 tract. The refinement included residual dipolar coupling (RDC) restraints. A radius of curvature, Rc, analysis was used to measure the overall bending in the molecule. A plot of the helical axis reference points showed a sigmoidal shape indicating a discontinuity at the central TpA step in the overall curvature. Because of the length of the 16mer it was possible to accurately measure Rc for the two halves of the molecule centered about the TpA step. The Rc for the overall molecule (Rc=128 nm) is large, i.e. a low overall magnitude of global bending, whereas the Rc for the two halves of the molecule (Rc=72 nm) is small indicating a much larger magnitude of localized bending. However the direction of bending in the two halves is in partial opposition leading to cancellation of the overall bending. This indicates that TnAn-containing DNA sequences are strongly and multiply bent locally, but are nearly straight globally which is consistent with PAGE results. The RDC refined structure lacked anomalous features present in NOE-only structures indicating the RDC and NOE measurements have a different sensitivity to conformational dynamics at the central TpA step. Because of its increased length and refinement using RDC restraints, the structure of the 16mer reported here provides new insight into the structural origins of the enigmatic PAGE behavior of AnTn and TnAn tracts and the large amplitude, slow base dynamics observed at TpA steps.

Buchko GW, CS Tung, K Mcateer, NG Isern, LD Spicer, and MA Kennedy. 2001. "DNA-XPA Interactions: A 31P NMR Study of dCCAATAACC Association With the Minimal DNA-Binding Domain (M98-F219) of the Nucleotide Excision Repair Protein XPA." Nucleic Acids Research 29(12):2635-2643. Abstract XPA is a central protein component of nucleotide excision repair (NER), playing an important role in recognizing DNA damage and orchestrating other proteins into position to remove the damage. Recent chemical shift mapping experiments with the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) suggests that a basic cleft located in the loop-rich subdomain plays a role in DNA binding.

Buchko GW, NG Isern, LD Spicer, and MA Kennedy. 2001. "Human Nucleotide Excision Repair Protein XPA: NMR Spectroscopic Studies of an XPA Fragment Containing the ERCC1-Binding Region and the Minimal DNA-Binding Domain (M59-F219)." Mutation Research 486(1):1-10. Abstract XPA is a central protein component of nucleotide excision repair (NER), a ubiquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genome.

Daughdrill GW, J Ackerman, NG Isern, MV Botuyan, CH Arrowsmith, MS Wold, and DF Lowry. 2001. "The Weak Interdomain Coupling Observed in the 70 kDa Subunit of Human Replication Protein A is Unaffected by ssDNA Binding." Nucleic Acids Research 29(15):3270-3276. Abstract Replication protein A (RPA) is a heterotrimeric, multifunctional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70-protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA701-3261). Correlation analysis of the amide 1H and 15 N chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA701-326 with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA701-326, indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA701-326 and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA701-68).