bluuebunny/crossref_metadata_2025 huggingface dataset:
The crystal structure of the mouse glandular kallikrein‐13 (prorenin converting enzyme)
Authors: Timm David E.
Published: February 2009

A crystal structure of the serine protease, mouse glandular kallikrein 13 (mGK‐13) has been determined at 2.6‐Å resolution. This enzyme, isolated from the mouse submandibular gland, is also known as prorenin‐converting enzyme and cleaves submandibular gland Ren‐2 prorenin to yield active renin. The mGK‐13 structure is similar to other members of the mammalian serine protease family, having five conserved disulfide bonds and an active site located in the cleft between two β‐barrel domains. The mGK‐13 structure reveals for the first time an ordered kallikrein loop conformation containing a short 310 helix. This loop is disordered in the related porcine pancreatic kallikrein and rat submandibular tonin structures. The kallikrein loop is in close spatial proximity to the active site and is also involved in a dimeric arrangement of mGK‐13. The catalytic specificity of mCK‐13 for Ren‐2 prorenin was studied by modeling a prorenin‐derived peptide into the active site of mGK‐13. This model emphasizes two electronegative substrate specificity pockets on the mGK‐13 surface, which could accommodate the dibasic P2 and P1 residues at the site of prorenin cleavage by mGK‐13.
Structure‐based subsite specificity mapping of human cathepsin D using statine‐based inhibitors
Authors: Majer Pavel , Collins Jack R. , Gulnik Sergei V. , Erickson John W.
Published: February 2009

Human cathepsin D is a lysosomal aspartic protease that has been implicated in breast cancer metastasis and Alzheimer's disease. Based on a crystal structure of a human cathepsin D‐pepstatin A complex, a series of statine‐containing inhibitors was designed, synthesized, and tested for inhibitory activity toward the enzyme in vitro. The compounds were modified systematically at individual positions (P4, P3, P2, P1, and P'2) with the aim of mapping the cathepsin D subsite preferences. The experimentally obtained SAR data were correlated on the basis of molecular modeling. Side‐chain preferences for the peptidomimetic inhibitors differed from those found previously using peptide substrates (Scarborough PE et al., 1993, Protein Sci 2:264‐276). In addition, the effects of single side‐chain modifications were often nonadditive. Structure‐activity relationships, modeling, and thermodynamic analysis indicated that entropy plays a major stabilizing role in inhibitor binding to cathepsin D.
Structural studies of receptor binding by cholera toxin mutants
Authors: Merritt Ethan A. , Sarfaty Steve , Hol WIM G.J. , Jobling Michael G. , Holmes Randall K. , Chang T. , Hirst Timothy R.
Published: February 2009

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B‐pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain.

We now report the structure determination and 2.3‐Å refinement of the CTB mutant Gly 33 → Arg complexed with the Gm1 oligosaccharide, as well as the 2.2‐Å refinement of a Gly 33 → Asp mutant of the closely related Escherichia coli heat‐labile enterotoxin B‐pentamer (LTB). Two of the five receptor binding sites in the Gly 33 → Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 Å) determination and refinement of the wild‐type CTB:GM1oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild‐type and Gly 33 → Arg mutant complexes.

The protein conformation observed for the binding‐deficient Gly 33 → Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33 → Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self‐affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.
Epitope and mimotope for an antibody to the Na, K‐ATPase
Authors: Pacholczyk Tadeusz , Sweadner Kathleen J.
Published: February 2009

The epitope of a monoclonal antibody specific for the α2 isoform of the Na,K‐ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N‐chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K‐ATPase N‐terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K‐ATPase. Full‐length cDNA for the Na,K‐ATPase was randomly fragmented and cloned into β‐galactosidase to create a λgt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N‐terminus. Chimeras of Na,K‐ATPase α1 and α2 narrowed down the epitope to 14‐19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K‐ATPase α2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or β‐galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N‐terminus, where the Na,K‐ATPase isofonns differ most.
Mutational analysis of the BPTI folding pathway: II. Effects of aromatic → leucine substitutions on folding kinetics and thermodynamics
Authors: Zhang Jian‐Xin , Goldenberg David P.
Published: February 2009

The rates of the individual steps in the disulfide‐coupled folding and unfolding of eight BPTI variants, each containing a single aromatic to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide‐bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they had significantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically‐preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild‐type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.
Cloning, purification, and preliminary characterization by circular dichroism and NMR of a carboxyl‐terminal domain of the bacteriophage P22 scaffolding protein
Authors: Parker Matthew H. , Prevelige Peter E. , Jablonsky Michael , Casens Sherwood , Sampson Laura , Krishna N. Rama
Published: February 2009

Assembly of double‐stranded DNA viruses and bacteriophages involves the polymerization of several hundred molecules of coat protein, directed by an internal scaffolding protein. A 163‐amino acid carboxyl‐terminal fragment of the 303‐amino acid bacteriophage P22 scaffolding protein was cloned, over‐expressed, and purified. This fragment is active in procapsid assembly reactions in vitro. The circular dichroism spectrum of the fragment, as well as the 1D‐NMR and 15N‐1H HSQC spectra of the uniformly‐labeled protein, indicate that stable secondary structure elements are present. Determination of the three dimensional packing of these elements into the folded scaffolding protein fragment is underway. Structure‐based drug design targeted at structural proteins required for viral assembly may have potential as a therapeutic strategy.
Three‐dimensional structures and contexts associated with recurrent amino acid sequence patterns
Authors: Han Karen E , Bystroff Christopher , Baker David
Published: February 2009

We have used cluster analysis to identify recurring sequence patterns that transcend protein family boundaries. A subset of these patterns occur predominantly in a single type of local structure in proteins. Here we characterize the three‐dimensional structures and contexts in which these sequence patterns occur, with particular attention to the interactions responsible for their structural selectivity.
Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: Sequence analysis and mutagenesis identify receptor binding epitopes
Authors: Jerva L. Fred , Lolis Elias , Sullivan Gail
Published: February 2009

Murine macrophage inflammatory protein‐2 (MIP‐2), a member of the α‐ chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the α‐chemokines, such as interleukin‐8, gro‐α/MGSA, and neutrophil activating peptide‐2 (NAP‐2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP‐2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP‐2 exhibits a highly ordered secondary structure consistent with the α/β structures of other chemokines. Recombinant MIP‐2 is chemotactic for human and murine neutrophils and up‐regulates cell surface expression of Mac‐1. MIP‐2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP‐2 to the human types A and B IL‐8 receptors and the murine homologue of the IL‐8 receptor. MIP‐2 displays low‐affinity binding to the type A IL‐8 receptor (Kd >120 nM) and high‐affinity binding to the type B IL‐8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three‐dimensional structure of IL‐8 and sequence analysis of six chemokines (IL‐8, gro‐α, NAP‐2, ENA‐78, KC, and MIP‐2) that display highaffinity binding to the IL‐8 type B receptor are used to identify an extended N‐terminal surface that interacts with this receptor. Two mutants of MIP‐2 establish that this region is also involved in binding and activating the murine homologue of the IL‐8 receptor. Differences in the sequence between IL‐8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL‐8 for binding to the type A receptor.
Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A
Authors: Catanzano Francesca , Barone Guido , Graziano Giuseppe , Capasso Sante
Published: February 2009

Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature‐induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0‐6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 °C of denaturation temperature and 65 kJ mol −1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X‐ray three‐dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine‐2′‐monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.
Coupling backbone flexibility and amino acid sequence selection in protein design
Authors: Su Alyce , Mayo Stephen L.
Published: February 2009

Using a protein design algorithm that considers side‐chain packing quantitatively, the effect of explicit backbone motion on the selection of amino acids in protein design was assessed in the core of the streptococcal protein G β1 domain (Gβ1). Concerted backbone motion was introduced by varying Gβ1′s supersecondary structure parameter values. The stability and structural flexibility of seven of the redesigned proteins were determined experimentally and showed that core variants containing as many as 6 of 10 possible mutations retain native‐like properties. This result demonstrates that backbone flexibility can be combined explicitly with amino acid side‐chain selection and that the selection algorithm is sufficiently robust to tolerate perturbations as large as 15% of Gβ1's native supersecondary structure parameter values.
The membrane topology of the amino‐terminal domain of the red cell calcium pump
Authors: Castello Pablo R. , Flecha F. Luis González , Caride Ariel J. , ández Horacio N.Fern , Delfino José M. , Rossi JUAN P.F.C.
Published: February 2009

A systematic study of the membrane‐associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3‐(trifluorometnyl)‐3‐(m‐[ 125I]iodophenyl)‐diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane‐bound probe, [3H]bis‐phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light‐dependent labeling of an Mr 135,000‐140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS‐PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane‐embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N‐terminal domain of the Ca2+ pump. A labeled peptide of Mr 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino‐terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1‐322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with α‐helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N‐terminus of the Ca2+ pump.
The effect of denaturants on protein structure
Authors: Dunbar Jennifer , Yennawar Hemant P. , Banerjee Soojay , Luo Jiabin , Farber Gregory K.
Published: February 2009

Virtually all studies of the protein‐folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent around the protein can be altered. X‐ray crystallography is the obvious experimental technique to probe both types of changes. In this paper, we report the crystal structures of dihydrofolate reductase with urea and of ribonuclease A with guanidinium chloride. These two classic denaturants have similar effects on the native structure of the protein. The most important change that occurs is a reduction in the overall thermal factor. These structures offer a molecular explanation for the reduction in mobility. Although the reduction is observed only with the native enzyme in the crystal, a similar decrease in mobility has also been observed in the unfolded state in solution (Makhatadze G, Privalov PL. 1992. Protein interactions with urea and guanidinium chloride: A calorimetric study. J Mol Biol 226:491‐505).
Heteronuclear NMR assignments and secondary structure of the coiled coil trimerization domain from cartilage matrix protein in oxidized and reduced forms
Authors: Wiltscheck Ronald , Dames Sonja A. , Alexandrescu Andrei T. , Kammerer Richard A. , Schulthess Therese , Blommers MARCEL J.J. , Engel JŮRgen
Published: February 2009

The C‐terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43‐residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence‐specific assignments for 1H‐, 15N‐, and 13C‐NMR resonances have been obtained from 2D 1H NOESY and TOCSY spectra, and from 3D HNCA, 15N NOESY‐HSQC, and HCCH‐TOCSY spectra. A stretch of α‐helix encompassing five heptad repeats (35 residues) has been identified from intra‐chain HN‐HN and HN‐Hα NOE connectivities, 3 JHNHα coupling constants, and chemical shift indices. The α‐helix begins immediately downstream of inter‐chain disulfide bonds between residues Cys 5 and Cys 7, and extends to near the C‐terminus of the molecule. The threefold symmetry of the molecule is maintained when the inter‐chain disulfide bonds that flank the N‐terminus of the coiled coil are reduced. Residues Ile 21 through Glu 36 show conserved chemical shifts and NOE connectivities, as well as strong protection from solvent exchange in the oxidized and reduced forms of the protein. By contrast, residues Ile 10 through Val 17 show pronounced chemical shift differences between the oxidized and reduced protein. Strong chemical exchange NOEs between HN resonances and water indicate solvent exchange on time scales faster than 10 s, and suggests a dynamic fraying of the N‐terminus of the coiled coil upon reduction of the disulfide bonds. Possible roles for the disulfide crosslinks of the oligomerization domain in the function of cartilage matrix protein are proposed.
Determination of the binding frame of the chaperone SecB within the physiological ligand oligopeptide‐binding protein
Authors: Smith Virginia F. , Randall Linda L. , Hardy SIMON J. S.
Published: February 2009

Chaperone proteins demonstrate the paradoxical ability to bind ligands rapidly and with high affinity but with no apparent sequence specificity. To learn more about this singular property, we have mapped the binding frame of the chaperone SecB from E. coli on the oligopeptide‐binding protein. Similar studies performed on the maltose‐binding and galactose‐binding proteins revealed centrally positioned binding frames of ∼160 aminoacyl residues. The work described here shows that OppA, which is significantly longer than the previously studied ligands, has a binding frame that covers 460 amino acids, nearly the entire length of the protein. We propose modes of binding to account for the data.
Dimethyl sulfoxide binding to globular proteins: A nuclear magnetic relaxation dispersion study
Authors: Jóhannesson Haukur , Denisov Vladimir P. , Halle Bertil
Published: February 2009

The 2H magnetic relaxation dispersion (NMRD) technique was used to characterize interactions of dimethyl sulfoxide (DMSO) with globular proteins. A difference NMRD experiment involving the N‐acetylglucosamine trisaccharide inhibitor, demonstrated that the DMSO 2H NMRD profile in lysozyme solution is due to a single DMSO molecule bound in the active cleft, with a molecular order parameter of 0.47 + 0.05 and a residence time in the range 10 ns to 5 ms. With the aid of transverse 2H relaxation data, the upper bound of the residence time was further reduced to 100 μs. A 1H shift titration experiment was also performed, yielding a binding constant of 2.3 + 0.3 M−1 at 27 °C. In contrast to lysozyme, no DMSO dispersion was observed for bovine pancreatic trypsin inhibitor (BPTI), indicating that a stable DMSO‐protein complex requires a cleft of appropriate geometry in addition to hydrogen‐bond and hydrophobic interactions.
Circular dichroism determination of class I MHC‐peptide equilibrium dissociation constants
Authors: Morgan Chantal S. , Holton James M. , Olafson Barry D. , Bjorkman Pamela J. , Mayo Stephen L.
Published: February 2009

Class I major histocompatibility complex (MHC) molecules bind peptides derived from degraded proteins for display to T cells of the immune system. Peptides bind to MHC proteins with varying affinities, depending upon their sequence and length. We demonstrate that the thermal stability of the MHC‐peptide complex depends directly on peptide binding affinity. We use this correlation to develop a convenient method to determine peptide dissociation constants by measuring MHC‐peptide complex stability using thermal denaturation profiles monitored by circular dichroism.
Structural motifs at protein‐protein interfaces: Protein cores versus two‐state and three‐state model complexes
Authors: Tsai Chung‐Jung , Xu Dong , Nussinov Ruth
Published: February 2009

The general similarity in the forces governing protein folding and protein‐protein associations has led us to examine the similarity in the architectural motifs between the interfaces and the monomers. We have carried out extensive, all‐against‐all structural comparisons between the single‐chain protein structural dataset and the interface dataset, derived both from all protein‐protein complexes in the structural database and from interfaces generated via an automated crystal symmetry operation. We show that despite the absence of chain connections, the global features of the architectural motifs, present in monomers, recur in the interfaces, a reflection of the limited set of the folding patterns. However, although similarity has been observed, the details of the architectural motifs vary. In particular, the extent of the similarity correlates with the consideration of how the interface has been formed. Interfaces derived from two‐state model complexes, where the chains fold cooperatively, display a considerable similarity to architectures in protein cores, as judged by the quality of their geometric superposition. On the other hand, the three‐state model interfaces, representing binding of already folded molecules, manifest a larger variability and resemble the monomer architecture only in general outline. The origin of the difference between the monomers and the three‐state model interfaces can be understood in terms of the different nature of the folding and the binding that are involved. Whereas in the former all degrees of freedom are available to the backbone to maximize favorable interactions, in rigid body, three‐state model binding, only six degrees of freedom are allowed. Hence, residue or atom pair‐wise potentials derived from protein‐protein associations are expected to be less accurate, substantially increasing the number of computationally acceptable alternate binding modes (Finkelstein et al., 1995).
Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP‐dependent protein kinase
Authors: Gibson Robin M. , Ji‐Buechler Ying , Taylor Susan S.
Published: February 2009

Two classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP‐dependent protein kinase (cAPK), the heat‐stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits. Basic sites on C, previously identified as important for R/C interaction in yeast TPKI and corresponding to Lys213, Lys217, and Lys189 in murine Cα, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI. rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI. This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994). Thus, the C‐subunit employs two distinct electrostatic surfaces to achieve high‐affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft. Unlike TPK1, mutation of Lys189 had no effect. The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C. Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations, rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C. The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.
NMR chemical shift perturbation mapping of dna binding by a zinc‐finger domain from the yeast transcription factor ADR1
Authors: Schmiedeskamp Mia , Rajagopal Ponni , Klevit Rachel E.
Published: February 2009

Mutagenesis studies have revealed that the minimal DNA‐binding domain of the yeast transcription factor ADR1 consists of two Cys2‐His2 zinc fingers plus an additional 20 residues proximal and N‐terminal to the fingers. We have assigned NMR 1'H, 15N, and 13C chemical shifts for the entire minimal DNA‐binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single‐finger constructs, and 13Cα chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein‐DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N‐terminus of the finger 2 helix is affected. Furthermore, residues N‐terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein‐DNA interface. A striking correspondence is observed between the protein‐DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis.
Conformational studies of the N‐terminal lipid‐associating domain of human apolipoprotein C‐I by CD and 1H NMR spectroscopy
Authors: Rozek Annett , Buchko Garry W. , Kanda Patrick , Cushley Robert J.
Published: February 2009

A peptide comprising the N‐terminal 38 residues of human apolipoprotein C‐I (apoC‐I(1‐38)) was synthesized using solid‐phase methods and its solution conformation studied by CD and 1H NMR spectroscopy. The CD data indicate that apoC‐I(1‐38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS‐bound apoC‐I(1‐38) was calculated from 464 NOE‐based distance restraints using distance geometry methods. ApoC‐I(1‐38) adopts a helical structure between residues V4 and K30 and an extended C‐terminus from Q31 when associated with SDS. The region K12‐G15 undergoes slow conformational exchange as indicated by above‐average amide resonance linewidths, large temperature coefficients, and fast exchange (<2 h) of backbone amide protons with deuterium. The mobility of K12‐G15 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC‐I(1‐38) is curved toward its hydrophobic face with bends of 125°, centered at K12/E13, and 150°, centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, I26, and I29, with the amphiphile SDS. Based on our present structural definition of apoC‐I(1‐38) and the previously obtained structure of the fragment apoC‐I(35‐53), we propose the secondary structure of intact apolipoprotein C‐I.
Solution structure of αtα, a helical hairpin peptide of de novo design
Authors: Fezoui Youcef , Connolly Peter J. , Osterhout John J.
Published: February 2009

αtα is a 38‐residue peptide designed to adopt a helical hairpin conformation in solution (Fezoui Y, Weaver DL, Osterhout JJ, 1995, Protein Sci 4286‐295). A previous study of the carboxylate form of αtα by CD and two‐dimensional NMR indicated that the peptide was highly helical and that the helices associated in approximately the intended orientation (Fezoui Y, Weaver DL, Osterhout JJ, 1994, Proc Natl Acad Sci USA 91:3675‐3679). Here, the solution structure of αtα as determined by two‐dimensional NMR is reported. A total of 266 experimentally derived distance restraints and 20 dihedral angle restraints derived from J‐couplings were used. One‐hundred initial structures were generated by distance geometry and refined by dynamical simulated annealing. Twenty‐three of the lowest‐energy structures consistent with the experimental restraints were analyzed. The results presented here show that αtα is comprised of two associating helices connected by a turn region.
Probing minimal independent folding units in dihydrofolate reductase by molecular dissection
Authors: Gegg Colin V. , Bowers Katherine E. , Matthews C. Robert
Published: February 2009

Molecular dissection was employed to identify minimal independent folding units in dihydrofolate reductase (DHFR) from Escherichia coli. Eight overlapping fragments of DHFR, spanning the entire sequence and ranging in size from 36 to 123 amino acids, were constructed by chemical cleavage. These fragments were designed to examine the effect of tethering multiple elements of secondary structure on folding and to test if the secondary structural domains represent autonomous folding units. CD and fluorescence spectroscopy demonstrated that six fragments containing up to a total of seven α‐helices or β‐strands and, in three cases, the adenine binding domain (residues 37‐86), are largely disordered. A stoichiometric mixture of the two fragments comprising the large discontinuous domain, 1‐36 and 87‐159, also showed no evidence for folding beyond that observed for the isolated fragments. A fragment containing residues 1‐107 appears to have secondary and tertiary structure; however, spontaneous self‐association made it impossible to determine if this structure solely reflects the behavior of the monomeric form. In contrast, a monomeric fragment spanning residues 37‐159 possesses significant secondary and tertiary structure. The urea‐induced unfolding of fragment 37‐159 in the presence of 0.5 M ammonium sulfate was found to be a well‐defined, two‐state process. The observation that fragment 37‐159 can adopt a stable native fold with unique, aromatic side‐chain packing is quite striking because residues 1‐36 form an integral part of the structural core of the full‐length protein.
Structural analysis of three His32 mutants of DsbA: Support for an electrostatic role of His32 in DsbA stability
Authors: Guddat Luke W. , Bardwell James C. A. , Glockshuber Rudi , Huber‐Wunderlich Martina , Zander Thomas , Martin Jennifer L.
Published: February 2009

DsbA, a 21‐kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys30‐Pro31‐His32‐Cys33). Unlike most protein disulfides, the active‐site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH.

His32, one of the two residues between the active‐site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of ‐5‐7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild‐type protein.

Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 variants are very similar to the wild‐type oxidized structure, including conservation of solvent atoms near the active‐site residue, Cys30. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.
pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pKa values
Authors: Singer Alex U. , Forman‐Kay Julie D.
Published: February 2009

Electrostatic interactions in a complex of the phospholipase C‐γ, C‐terminal SH2 domain with a high‐affinity binding phosphopeptide representing the sequence around Tyr 1021 of the β platelet‐derived growth factor receptor were studied by pKa determination of various titratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His βD4) and the phosphotyrosine (pTyrj phosphate group show pKa values significantly lower than average for these residue types in proteins. The reduced pKa of these two groups is due to the proximity of the highly positively charged pTyr binding pocket. The unusual pK, of His PD4 is also due to burial from solvent in a hydrogen‐bonding network that appears necessary for the positioning of arginine residues involved in pTyr binding. Mutation of the analogous histidine in other SH2 domains has been shown to abrogate pTyr binding. In addition to these large shifts in pKa, values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C‐terminus of the the second helix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domain‐phosphopeptide complex confirms previous descriptions of the roles of different arginines in the structure and function of this protein.
Empirical parameterization of a model for predicting peptide helix/coil equilibrium populations
Authors: Andersen Niels H. , Tong Hui
Published: February 2009

A modification of the Lifson‐Roig formulation of helix/coil transitions is presented; it (1) incorporates end‐capping and coulombic (salt bridges, hydrogen bonding, and side‐chain interactions with charged termini and the helix dipole) effects, (2) helix‐stabilizing hydrophobic clustering, (3) allows for different inherent termination probabilities of individual residues, and (4) differentiates helix elongation in the first versus subsequent turns of a helix. Each residue is characterized by six parameters governing helix formation. The formulation of the conditional probability of helix initiation and termination that we developed is essentially the same as one presented previously (Shalongo W, Stellwagen, E. 1995. Protein Sci 41161‐1166) and nearly the mathematical equivalent of the new capping formulation incorporated in the model presented by Rohl et al. (1996. Protein Sci 5:2623‐2637). Side‐chain/side‐chain interactions are, in most cases, incorporated as context dependent modifications of propagation rather than nucleation parameters. An alternative procedure for converting [θ]221 values to experimental fractional helicities (〈fH〉) is presented. Tests of the program predictions suggest this method may have some advantages both for designed peptides and for the analysis of secondary structure preferences that could drive the formation of molten‐globule intermediates on protein folding pathways. The model predicts the fractional helicity of 385 peptides with a root‐mean‐square deviation (RMSD) of 0.050 and locates (with precise definition of the termini in many cases) helices in proteins as well as competing methods. The propagation and nucleation parameters were derived from NMR data and from the CD data for a 79 peptide “learning set” for which an excellent fit resulted (RMSD = 0.0295). The current set of parameter corrections for capping boxes, helix dipole interactions, and side‐chain/side‐chain interactions (coulombic, hydrogen bonding, and hydrophobic clustering), although still under development, provide a significant improvement in both helix/coil equilibrium prediction for peptides and helix location in protein sequences. This is clearly evident in the rms deviations between CD measures and calculated values of fractional helicity for different classes of peptides before and after applying the corrections: for peptides lacking capping boxes and i/i + 3 and i/i + 4 side‐chain/side‐chain interactions RMSD = 0.044 (n = 164) versus RMSD = 0.054 (0.172 without the corrections, n = 221) for peptides that required context‐dependent corrections of the parameters. If we restrict the analysis to N‐acylated peptides with helix stabilizing side‐chain/side‐chain interactions (including N‐capping boxes), the degree to which our corrections account for the stabilizing interaction can be judged from the change in helicity underestimation, (〈fH〉calc ‐ 〈fH〈CD): ‐0.15 + 0.10, which is reduced to ‐0.018 + 0.048 (n = 191) upon applying the corrections.
Determination of pKa values of the histidine side chains of phosphatidylinositol‐specific phospholipase C from Bacillus cereus by NMR spectroscopy and site‐directed mutagenesis
Authors: Liu Tun , Ryan Margret , Dahlquist Frederick W. , Griffith O. Hayes
Published: February 2009

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol‐specific phospholipase C (PI‐PLC). In this report, we present the first study of the pKa values of histidines of a PI‐PLC. All six histidines of Bacillus cereus PI‐PLC were studied by 2D NMR spectroscopy and site‐directed mutagenesis. The protein was selectively labeled with 13Cϵ1‐histidine. A series of 1H‐13C HSQC NMR spectra were acquired over a pH range of 4.0‐9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H‐13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild‐type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.
Increased production of low molecular weight recombinant proteins in Escherichia coli
Authors: Belagaje Rama M. , Reams Stephen G. , Ly Stan C. , Prouty Walter F.
Published: February 2009

A general method for obtaining high‐level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met‐Xaa‐protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N‐terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high‐level production of human insulin‐like growth factor‐1, human proinsulin, and their analogs. Furthermore, highly produced insulin‐like growth factor‐1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.
Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation
Authors: Dominici Paola , Moore Patrick S. , Castellani Silvia , Bertoldi Mariarita , Voltattorni Carla Borri
Published: February 2009

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site‐directed mutagenesis. Compared to the wild‐type enzyme, the resultant C111A and C111 S mutant enzymes exhibit kcat values of about 50% and 15%, respectively, at pH 6.8, while the Km values remain relatively unaltered for L‐3,4‐dihydroxyphenylalanine (L‐Dopa) and L‐5‐hydroxytryptophan (L‐5‐HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co‐enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys‐111→Ala mutant displays a reduced affinity for pyridoxal 5′‐phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D‐Dopa and the bound co‐enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild‐type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild‐type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420 → 390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys‐111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Bom Voltattorni C. 1991. Eur J Biochem 201:393‐397). Moreover, our findings provide evidence that Cys‐111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active‐site conformation.
Crystallization and preliminary X‐ray crystallographic properties of Hsc20, a J‐motif co‐chaperone protein from Escherichia coli
Authors: Cupp‐Vickery Jill R. , Vickery Larry E.
Published: February 2009

Hsc20 is a 20‐kDa auxiliary protein that functions with the molecular chaperone Hsc66 in Escherichia coli. Crystals of Hsc20 suitable for X‐ray diffraction analysis were grown using the hanging drop vapor diffusion technique in polyethylene glycol 400 containing dioxane as an additive to slow growth. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions α = 125.4 Å, b = 71.9 Å, c = 68.8 Å, and β = 97.0°. The crystals diffract to a minimum d‐spacing of ∼2.5 Å resolution, and a native data set was collected to 2.7 Å. The results of a self‐rotation function analysis revealed threefold symmetry, suggesting three molecules of Hsc20 in the asymmetric unit and, hence, 12 molecules in the unit cell; this corresponds to a Vm value of 2.6 Å3/Da and a solvent content of ∼53% in the crystals. Structure determination by isomorphous replacement is in progress.
Sequence analysis of the AAA protein family
Authors: Beyer Andreas
Published: February 2009

The AAA protein family, a recently recognized group of Walker‐type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so‐called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra‐relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases.
Ideal architecture of residue packing and its observation in protein structures
Authors: Raghunathan G. , Jernigan R. L.
Published: February 2009

A simple model of sphere packing has been investigated as an ideal model for long‐range interactions for the packing of non‐bonded residues in protein structures. By superposing all residues, the geometry of packing around a central residue is investigated. It is found that all residues conform almost perfectly to this lattice model for sphere packing when a radius of 6.5 Å is used to define non‐bonded (virtual) interacting residues. Side‐chain positions with respect to sequential backbone segments are relatively regular as well. This lattice can readily be used in conformation simulations to reduce the conformational space.