bluuebunny/crossref_metadata_2025 huggingface dataset:
Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles
Authors: Sanghera Narinder , Pinheiro Teresa J.T.
Published: February 2009

Binding of native cyt c to L‐PG micelles leads to a partially unfolded conformation of cyt c. This micelle‐bound state has no stable tertiary structure, but remains as α‐helical as native cyt c in solution. In contrast, binding of the acid‐unfolded cyt c to L‐PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L‐PG micelles. Far‐ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle‐associated helical state (HL) has a native‐like α‐helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near‐UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid‐unfolded cyt c was investigated by stopped‐flow tryptophan fluorescence. Formation of HL from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle‐induced unfolding of cyt c occurs at a rate ∼0.1 s∽1, which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid‐unfolded cyt c with L‐PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.
Amino acid repeat patterns in protein sequences: Their diversity and structural‐functional implications
Authors: Katti Mukund V. , Sami‐Subbu R. , Ranjekar Prabhakar K. , Gupta Vidya S.
Published: February 2009

All the protein sequences from SWISS‐PROT database were analyzed for occurrence of single amino acid repeats, tandem oligo‐peptide repeats, and periodically conserved amino acids. Single amino acid repeats of glutamine, serine, glutamic acid, glycine, and alanine seem to be tolerated to a considerable extent in many proteins. Tandem oligo‐peptide repeats of different types with varying levels of conservation were detected in several proteins and found to be conspicuous, particularly in structural and cell surface proteins. It appears that repeated sequence patterns may be a mechanism that provides regular arrays of spatial and functional groups, useful for structural packing or for one to one interactions with target molecules. To facilitate further explorations, a database of Tandem Repeats in Protein Sequences (TRIPS) has been developed and is available at URL: http://www.ncl‐india.org/trips.
Molecular simulation study of cooperativity in hydrophobic association
Authors: Czaplewski Cezary , Rodziewicz‐Motowidlo Sylwia , Liwo Adam , Ripoll Daniel R. , Wawak Ryszard J. , Scheraga Harold A.
Published: February 2009

To investigate the cooperativity of hydrophobic interactions, the potential of mean force of two‐ and three‐molecule methane clusters in water was determined by molecular dynamics simulations using two methods: umbrella‐sampling with the weighted histogram analysis method and thermodynamic integration. Two water models, TIP3P and TIP4P, were used, while each methane molecule was modeled as a united atom. It was found that the three‐body potential of mean force is not additive, i.e., it cannot be calculated as a sum of two‐body contributions, but requires an additional three‐body cooperative term. The cooperative term, which amounts to only about 10% of the total hydrophobic association free energy, was found to increase the strength of hydrophobic association; this finding differs from the results of earlier Monte Carlo studies with the free energy perturbation method of Rank and Baker (1997). As in the work of Rank and Baker, the solvent contribution to the potential of mean force was found to be well approximated by the molecular surface of two methane molecules. Moreover, we also found that the cooperative term is well represented by the difference between the molecular surface of the three‐methane cluster and those of all three pairs of methane molecules. In addition, it was found that, while there is a cooperative contribution to the hydrophobic association free energy albeit a small one, the errors associated with the use of pairwise potentials are comparable to or larger than this contribution.
Assisted folding of D‐glyceraldehyde‐3‐phosphate dehydrogenase by trigger factor
Authors: Huang Guo‐Chang , Li Zhen‐Yu , Zhou Jun‐Mei , Fischer Gunter
Published: February 2009

The Escherichia coli trigger factor is a peptidyl‐prolyl cis‐trans isomerase that catalyzes proline‐limited protein folding extremely well. Here, refolding of D‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either long‐or short‐term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down‐turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced αLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor.
Topography of a 2.0 Å structure of α1‐antitrypsin reveals targets for rational drug design to prevent conformational disease
Authors: Elliott Peter R. , Pei Xue Y. , Dafforn Timothy R. , Lomas David A.
Published: February 2009

Members of the serpin family of serine proteinase inhibitors play important roles in the inflammatory, coagulation, fibrinolytic, and complement cascades. An inherent part of their function is the ability to undergo a structural rearrangement, the stressed (S) to relaxed (R) transition, in which an extra strand is inserted into the central A β‐sheet. In order for this transition to take place, the A sheet has to be unusually flexible. Malfunctions in this flexibility can lead to aberrant protein linkage, serpin inactivation, and diseases as diverse as cirrhosis, thrombosis, angioedema, emphysema, and dementia. The development of agents that control this conformational rearrangement requires a high resolution structure of an active serpin. We present here the topology of the archetypal serpin α1‐antitrypsin to 2 Å resolution. This structure allows us to define five cavities that are potential targets for rational drug design to develop agents that will prevent conformational transitions and ameliorate the associated disease.
NMR structure of a concatemer of the first and second ligand‐binding modules of the human low‐density lipoprotein receptor
Authors: Kurniawan Nyoman D. , Atkins Annette R. , Bieri Stephan , Brown Catherine J. , Kroon Paulus A. , Smith Ross , Brereton Ian M.
Published: February 2009

The ligand‐binding domain of the human low‐density lipoprotein receptor consists of seven modules, each of 40–45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB1‐2) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three‐dimensional structure of LB1‐2 has now been solved using two‐dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random‐coil NH and Hα chemical shifts and the low ϕ and ψ angle order parameters of the four‐residue linker suggest that it has considerable flexibility. The family of LB1‐2 structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand‐binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.
Soft metal ions, Cd(II) and Hg (II), induce triple‐stranded α‐helical assembly and folding of a de novo designed peptide in their trigonal geometries
Authors: Li Xiangqun , Suzuki Kazuo , Kashiwada Ayumi , Hiroaki Hidekazu , Kohda Daisuke , Tanaka Toshiki
Published: February 2009

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native‐like, parallel triple‐stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile 18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple‐stranded α‐helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple‐stranded α‐helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 ± 0.8 μ for Cd(II). The triple‐stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl− ions. The 113Cd NMR resonance was connected with the βH protons of the cysteine residue by 1H‐113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal‐planar complex. Hg(II) also induced the assembly of the peptide into a triple‐stranded α‐helical bundle below the Hg (II)/peptide ratio of 1/3. With excess Hg(II), however, the α‐helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.
Bovine β‐lactoglobulin: Interaction studies with palmitic acid
Authors: Ragona Laura , Zetta Lucia , Fogolari Federico , Molinari Henriette , Pérez Dolores M. , Puyol Pilar , Kruif Kees De , Löhr Frank , Rüterjans Heinz
Published: February 2009

Bovine β‐lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure‐function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one‐, two‐, and three‐dimensional NMR spectroscopy in the pH range 8.4‐2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4–5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.
The disulfide bond pattern of catrocollastatin C, a disintegrin‐like/cysteine‐rich protein isolated from Crotalus atrox venom
Authors: Calvete Juan J. , Moreno‐Murciano M. Paz , Sanz Libia , Jurgens Michael , Schrader Michael , Raida Manfred , Benjamin David C. , Fox Jay W.
Published: February 2009

The disulfide bond pattern of catrocollastatin‐C was determined by N‐terminal sequencing and mass spectrometry. The N‐terminal disintegrin‐like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin‐like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn‐metalloproteinases (SVMPs). The C‐terminal cysteine‐rich domain of catrocollastatin‐C contains five disulfide bonds between nearest‐neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin‐like and cysteine‐rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.
Structure of a protein G helix variant suggests the importance of helix propensity and helix dipole interactions in protein design
Authors: Strop Pavel , Marinescu Andrei M. , Mayo Stephen L.
Published: February 2009

Six helix surface positions of protein G (Gβ1) were redesigned using a computational protein design algorithm, resulting in the five fold mutant Gβ1m2. Gβ1m2 is well folded with a circular dichroism spectrum nearly identical to that of Gβ1, and a melting temperature of 91 °C, ∼6°C higher than that of Gβ1. The crystal structure of Gβ1m2 was solved to 2.0 Å resolution by molecular replacement. The absence of hydrogen bond or salt bridge interactions between the designed residues in Gβ1m2 suggests that the increased stability of Gβ1m2 is due to increased helix propensity and more favorable helix dipole interactions.
Charge–charge interactions influence the denatured state ensemble and contribute to protein stability
Authors: Nick Pace C. , Alston Roy W. , Shaw Kevin L.
Published: February 2009

Several recent studies have shown that it is possible to increase protein stability by improving electrostatic interactions among charged groups on the surface of the folded protein. How–ever, the stability increases are considerably smaller than predicted by a simple Coulomb's law calculation, and in some cases, a charge reversal on the surface leads to a decrease in stability when an increase was predicted. These results suggest that favorable charge–charge interactions are important in determining the dena–tured state ensemble, and that the free energy of the denatured state may be decreased more than that of the native state by reversing the charge of a side chain. We suggest that when the hydrophobic and hydrogen bonding interactions that stabilize the folded state are disrupted, the unfolded polypeptide chain rearranges to com–pact conformations with favorable long–range electrostatic inter–actions. These charge–charge interactions in the denatured state will reduce the net contribution of electrostatic interactions to protein stability and will help determine the denatured state ensemble. To support this idea, we show that the denatured state ensemble of ribonuclease Sa is considerably more compact at pH 7 where fa–vorable charge–charge interactions are possible than at pH 3, where unfavorable electrostatic repulsion among the positive charges causes an expansion of the denatured state ensemble. Further support is provided by studies of the ionic strength dependence of the sta–bility of charge–reversal mutants of ribonuclease Sa. These results may have important implications for the mechanism of protein folding.
Mesoscopic surfactant organization and membrane protein crystallization
Authors: Wiener Michael C. , Verkman Alan S. , Stroud Robert M. , Van Hoek Alfred N.
Published: February 2009

The paucity of detailed X–ray crystallographic struc–tures of integral membrane proteins arises from substantive technical obstacles in the overexpression of multimilligram quantities of protein, and in the crystallization of purified protein–detergent complexes (PDCs). With rare exception, crystal contacts within the lattice are mediated by protein‐protein interaction, and the detergent surrounding the protein behaves as a disordered solvent. The addition and use of surfactants that display mesoscopic self–assembly behavior in membrane protein crystallization experiments presents a novel alternative strategy. Well–ordered crystals of the water channel human aquaporin–1 (hAQP1) that diffract to 4 Å resolution have been obtained with this approach.
Recombinant decorsin: Dynamics of the RGD recognition site
Authors: Krezel Andrzej M. , Ulmer Jana S. , Wagner Gerhard , Lazarus Robert A.
Published: February 2009

Decorsin is an antagonist of integrin αIIbβ3 and a potent platelet aggregation inhibitor. A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli. The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. The protein was purified by size‐exclusion filtration of the periplasmic contents followed by reversed‐phase high‐performance liquid chromatography. Purified recombinant decorsin was found to be indistinguishable from leech‐derived decorsin based on amino acid composition, mass spectral analysis, and biological activity assays. Complete sequential assignments of 1H and proton bound 13C resonances were established. Stereospecific assignments of 21 of 25 nondegenerate β‐methylene groups were determined. The RGD adhesion site recognized by integrin receptors was found at the apex of a most exposed hairpin loop. The dynamic behavior of decorsin was analyzed using several independent NMR parameters. Although the loop containing the RGD sequence is the most flexible one in decorsin, the conformation of the RGD site itself is more restricted than in other proteins with similar activities.
Solution structure of a zinc substituted eukaryotic rubredoxin from the cryptomonad alga Guillardia theta
Authors: Schweimer Kristian , Hoffmann Silke , Rösch Paul , Sticht Heinrich , Wastl Jürgen , Maier Uwe G.
Published: February 2009

The rubredoxin from the cryptomonad Guillardia theta is one of the first examples of a rubredoxin encoded in a eukaryotic organism. The structure of a soluble zinc‐substituted 70‐residue G. theta rubredoxin lacking the membrane anchor and the thylakoid targeting sequence was determined by multidimensional heteronuclear NMR, representing the first three‐dimensional (3D) structure of a eukaryotic rubredoxin. For the structure calculation a strategy was applied in which information about hydrogen bonds was directly inferred from a long‐range HNCO experiment, and the dynamics of the protein was deduced from heteronuclear nuclear Overhauser effect data and exchange rates of the amide protons. The structure is well defined, exhibiting average root‐mean‐square deviations of 0.21 Å for the backbone heavy atoms and 0.67 Å for all heavy atoms of residues 7–56, and an increased flexibility toward the termini. The structure of this core fold is almost identical to that of prokaryotic rubredoxins. There are, however, significant differences with respect to the charge distribution at the protein surface, suggesting that G. theta rubredoxin exerts a different physiological function compared to the structurally characterized prokaryotic rubredoxins. The amino‐terminal residues containing the putative signal peptidase recognition/cleavage site show an increased flexibility compared to the core fold, but still adopt a defined 3D orientation, which is mainly stabilized by nonlocal interactions to residues of the carboxy‐terminal region. This orientation might reflect the structural elements and charge pattern necessary for correct signal peptidase recognition of the G. theta rubredoxin precursor.
Bypassing the kinetic trap of serpin protein folding by loop extension
Authors: Im Hana , Ahn Hee‐Young , Yu Myeong‐Hee
Published: February 2009

The native form of some proteins such as strained plasma serpins (serine protease inhibitors) and the spring‐loaded viral membrane fusion proteins are in a metastable state. The metastable native form is thought to be a folding intermediate in which conversion into the most stable state is blocked by a very high kinetic barrier. In an effort to understand how the spontaneous conversion of the metastable native form into the most stable state is prevented, we designed mutations of α1‐antitrypsin, a prototype serpin, which can bypass the folding barrier. Extending the reactive center loop of α1‐antitrypsin converts the molecule into a more stable state. Remarkably, a 30‐residue loop extension allows conversion into an extremely stable state, which is comparable to the relaxed cleaved form. Biochemical data strongly suggest that the strain release is due to the insertion of the reactive center loop into the major β‐sheet, A sheet, as in the known stable conformations of serpins. Our results clearly show that extending the reactive center loop is sufficient to bypass the folding barrier of α1‐antitrypsin and suggest that the constrain held by polypeptide connection prevents the conversion of the native form into the lowest energy state.
Chemical cross‐linking with thiol‐cleavable reagents combined with differential mass spectrometric peptide mapping—A novel approach to assess intermolecular protein contacts
Authors: Bennett Keiryn L. , Kussmann Martin , Mikkelsen Marie , Roepstorff Peter , Björk Per , Godzwon Magdalena , Sörensen Poul
Published: February 2009

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross‐linking with differential MALDI‐MS analyses. ParR dimers were modified in vitro with the thiol‐cleavable cross‐linker 3,3′‐dithio‐bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI‐MS peptide mapping. Comparison of the peptide maps obtained from digested cross‐linked ParR dimers in the presence and absence of a thiol reagent strongly supported a “head‐to‐tail” arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28‐IgG and CD80‐Fab were cross‐linked in vitro by DTSSP, characterized by non‐reducing SDS‐PAGE, digested in situ with trypsin and analyzed by MALDI‐MS peptide mapping (± thiol reagent). The data revealed the presence of an intermolecular cross‐link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28‐IgG and the receptor domain of CD80‐Fab. The strategy of chemical cross‐linking combined with differential MALDI‐MS peptide mapping ( thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.
Ligand binding and thermodynamic stability of a multidomain protein, calmodulin
Authors: Masino Laura , Martin Stephen R. , Bayley Peter M.
Published: February 2009

Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo‐domain follows a two‐state mechanism with relatively low δG°20 values of ˜2.7 (N‐domain) and ˜1.9 kcal/mol (C‐domain). The apo‐C‐domain is significantly unfolded at normal temperatures (20‐25 °C!. The greater affinity of the C‐domain for Ca2+ causes it to be more stable than the N‐domain at [Ca2+] ≤0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N‐ rather than the C‐domain, consistent with measured Mg2+ affinities.

For the intact protein (+Ca2+), the bimodal denaturation profiles can be analyzed to give two δG°20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.
Water‐protein interactions in the molten‐globule state of carbonic anhydrase b: An NMR spin‐diffusion study
Authors: Kutyshenko Victor P. , Cortijo Manuel
Published: February 2009

We have used the homonuclear Overhauser effect (NOE) to characterize a model protein: carbonic anhydrase B. We have obtained NOE difference spectra for this protein, centering the on‐resonance signals either at the methyl‐proton or at the water‐proton signals. The spin‐diffusion spectra obtained as a function of protein concentration and temperature provide direct evidence of much greater protein–water interaction in the molten‐globule state than in the native and denatured states. Furthermore, although the protein loses its gross tertiary structure in both the molten‐globule and denatured states, it remains almost as compact in its molten‐globule state as it is in the native state. The spin‐diffusion spectra, obtained as a function of a variable delay time after the saturation pulse, allowed us to measure the relaxation times of several types of proton in the solution. These spectra contain enough information to distinguish between those water molecules solvating the protein and the free ones present as bulk water.
Static light scattering studies of OmpF porin: Implications for integral membrane protein crystallization
Authors: Hitscherich Carl , Allaman Margaret , Wiencek John , Kaplan Jeffrey , Loll Patrick J.
Published: February 2009

Integral membrane proteins carry out some of the most important functions of living cells, yet relatively few details are known about their structures. This is due, in large part, to the difficulties associated with preparing membrane protein crystals suitable for X‐ray diffraction analysis. Mechanistic studies of membrane protein crystallization may provide insights that will aid in determining future membrane protein structures. Accordingly, the solution behavior of the bacterial outer membrane protein OmpF porin was studied by static light scattering under conditions favorable for crystal growth. The second osmotic virial coefficient (B22) was found to be a predictor of the crystallization behavior of porin, as has previously been found for soluble proteins. Both tetragonal and trigonal porin crystals were found to form only within a narrow window of B22 values located at approximately −0.5 to −2 × 10‐4 mol mL g‐2, which is similar to the “crystallization slot” observed for soluble proteins. The B22 behavior of protein‐free detergent micelles proved very similar to that of porin‐detergent complexes, suggesting that the detergent's contribution dominates the behavior of protein‐detergent complexes under crystallizing conditions. This observation implies that, for any given detergent, it may be possible to construct membrane protein crystallization screens of general utility by manipulating the solution properties so as to drive detergent B22 values into the crystallization slot. Such screens would limit the screening effort to the detergent systems most likely to yield crystals, thereby minimizing protein requirements and improving productivity.
Use of residue pairs in protein sequence‐sequence and sequence‐structure alignments
Authors: Jung Jongsun , Lee Byungkook
Published: February 2009

Two new sets of scoring matrices are introduced: H2 for the protein sequence comparison and T2 for the protein sequence–structure correlation. Each element of H2 or T2 measures the frequency with which a pair of amino acid types in one protein, k‐residues apart in the sequence, is aligned with another pair of residues, of given amino acid types (for H2) or in given structural states (for T2), in other structurally homologous proteins. There are four types, corresponding to the k‐values of 1 to 4, for both H2 and T2. These matrices were set up using a large number of structurally homologous protein pairs, with little sequence homology between the pair, that were recently generated using the structure comparison program SHEBA.

The two scoring matrices were incorporated into the main body of the sequence alignment program SSEARCH in the FASTA package and tested in a fold recognition setting in which a set of 107 test sequences were aligned to each of a panel of 3,539 domains that represent all known protein structures. Six procedures were tested; the straight Smith‐Waterman (SW) and FASTA procedures, which used the Blosum62 single residue type substitution matrix; BLAST and PSI‐BLAST procedures, which also used the Blosum62 matrix; PASH, which used Blosum62 and H2 matrices; and PASSC, which used Blosum62, H2, and T2 matrices. All procedures gave similar results when the probe and target sequences had greater than 30% sequence identity. However, when the sequence identity was below 30%, a similar structure could be found for more sequences using PASSC than using any other procedure. PASH and PSI‐BLAST gave the next best results.
The L6 membrane proteins—A new four‐transmembrane superfamily
Authors: Wright Mark D. , Rudy George B. , Ni Jian
Published: February 2009

L6, IL‐TMP, and TM4SF5 are cell surface proteins predicted to have four transmembrane domains. Previous sequence analysis led to their assignment as members of the tetraspanin superfamily. In this paper, we identify a new sequence (L6D) that is strikingly similar to L6, IL‐TMP, and TM4SF5. Analyses of these four sequences indicate that they are not significantly related to genuine tetraspanins, but instead constitute their own L6 superfamily.
The failure of simple empirical relationships to predict the viscosity of mixed aqueous solutions of guanidine hydrochloride and glucose has important implications for the study of protein folding
Authors: Sato Satoshi , Sayid Cosim J. , Raleigh Daniel P. , Raleigh Daniel P.
Published: February 2009

Viscosities of aqueous solutions of guanidine hydrochloride (GuHCl) were measured in the presence of varying amounts of glucose. At high concentrations of glucose or GuHCl, the measured viscosities showed significant deviation from the values computed using a method proposed by Tanford (1966, J Biol Chem 241:3228–3232). This method was originally derived to allow the calculation of the effects of buffer or low concentrations of salts and other additives on the density and viscosity of aqueous solutions of urea or GuHCl. Recently it has been used to estimate the viscosity of denaturant solutions that contain high concentrations of viscogens. Our results show that the extrapolation of this approach to solutions of highly concentrated viscous co‐solutes leads to significant errors. The implications for experimental studies of the viscosity dependence of conformational transitions in proteins is discussed.
Systematic mutational analysis of the active‐site threonine of HIV‐1 proteinase: Rethinking the “fireman's grip” hypothesis
Authors: Strisovsky Kvido , Tessmer Uwe , Langner Josmar , Konvalinka Jan , Kräusslich Hans‐Georg
Published: February 2009

Aspartic proteinases share a conserved network of hydrogen bonds (termed “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp‐Thr‐Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV‐1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp‐Thr‐Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV‐1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp‐Thr‐Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV‐1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp‐Thr‐Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV‐1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip,” Thr26 of HIV‐1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV‐1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild‐type HIV‐1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the “fireman's grip” interaction, was virtually inactive and was monomeric in solution at conditions where wild‐type PR exhibited a monomer‐dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV‐1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the “fireman's grip” is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the “fireman's grip” is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.
Understanding the sequence determinants of conformational switching using protein design
Authors: Dalal Seema , Regan Lynne
Published: February 2009

An important goal of protein design is to understand the forces that stabilize a particular fold in preference to alternative folds. Here, we describe an extension of earlier studies in which we successfully designed a stable, native‐like helical protein that is 50% identical in sequence to a predominantly β‐sheet protein, the B1 domain of Streptococcal IgG‐binding protein G. We report the characteristics of a series of variants of our original design that have even higher sequence identity to the B1 domain. Their properties illustrate the extent to which protein stability and conformation can be modulated through careful manipulation of key amino acid residues. Our results have implications for understanding conformational change phenomena of central biological importance and in probing the malleability of the sequence/structure relationship.
Immucillin‐H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange
Authors: Wang Fang , Miles Robert W. , Kicska Gregory , Nieves Edward , Schramm Vern L. , Angeletti Ruth Hogue
Published: February 2009

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI‐MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T‐cell immunodeficiency, while B‐cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T‐cell leukemia, to suppress the graft‐vs.‐host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin‐H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow‐exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin‐H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin‐H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132–152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29–49, 50–70, 81–98, and 112–124 decreased only in the complex with the transition state analogue. The peptide‐specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin‐H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.
Analysis of the internal motion of free and ligand‐bound human lysozyme by use of 15N NMR relaxation measurement: A comparison with those of hen lysozyme
Authors: Mine Shouhei , Ueda Tadashi , Hashimoto Yoshio , Imoto Taiji
Published: February 2009

Human lysozyme has a structure similar to that of hen lysozyme and differs in amino acid sequence by 51 out of 129 residues with one insertion at the position between 47 and 48 in hen lysozyme. The backbone dynamics of free or (NAG)3‐bound human lysozyme has been determined by measurements of 15N nuclear relaxation. The relaxation data were analyzed using the Lipari‐Szabo formalism and were compared with those of hen lysozyme, which was already reported (Mine S et al., 1999, J Mol Biol 286:1547–1565). In this paper, it was found that the backbone dynamics of free human and hen lysozymes showed very similar behavior except for some residues, indicating that the difference in amino acid sequence did not affect the behavior of entire backbone dynamics, but the folded pattern was the major determinant of the internal motion of lysozymes. On the other hand, it was also found that the number of residues in (NAG)3‐bound human and hen lysozymes showed an increase or decrease in the order parameters at or near active sites on the binding of (NAG)3, indicating the increase in picosecond to nanosecond. These results suggested that the immobilization of residues upon binding (NAG)3 resulted in an entropy penalty and that this penalty was compensated by mobilizing other residues. However, compared with the internal motions between both ligand‐bound human and hen lysozymes, differences in dynamic behavior between them were found at substrate binding sites, reflecting a subtle difference in the substrate‐binding mode or efficiency of activity between them.
High resolution refinement of β‐galactosidase in a new crystal form reveals multiple metal‐binding sites and provides a structural basis for α‐complementation
Authors: Juers Douglas H. , Jacobson Raymond H. , Wigley Dale , Zhang Xue‐Jun , Huber Reuben E. , Tronrud Dale E. , Matthews Brian W.
Published: February 2009

The unrefined fold of Escherichia coli β‐galactosidase based on a monoclinic crystal form with four independent tetramers has been reported previously. Here, we describe a new, orthorhombic form with one tetramer per asymmetric unit that has permitted refinement of the structure at 1.7 Å resolution. This high‐resolution analysis has confirmed the original description of the structure and revealed new details. An essential magnesium ion, identified at the active site in the monoclinic crystals, is also seen in the orthorhombic form. Additional putative magnesium binding sites are also seen. Sodium ions are also known to affect catalysis, and five putative binding sites have been identified, one close to the active site. In a crevice on the protein surface, five linked five‐membered solvent rings form a partial clathrate‐like structure. Some other unusual aspects of the structure include seven apparent cis‐peptide bonds, four of which are proline, and several internal salt‐bridge networks. Deep solvent‐filled channels and tunnels extend across the surface of the molecule and pass through the center of the tetramer. Because of these departures from a compact globular shape, the molecule is not well characterized by prior empirical relationships between the mass and surface area of proteins. The 50 or so residues at the amino terminus have a largely extended conformation and mostly lie across the surface of the protein. At the same time, however, segment 13–21 contributes to a subunit interface, and residues 29–33 pass through a “tunnel” formed by a domain interface. Taken together, the overall arrangement provides a structural basis for the phenomenon of α‐complementation.
Tryptophanyl fluorescence lifetime distribution of hyperthermophilic β‐glycosidase from molecular dynamics simulation: A comparison with the experimental data
Authors: Bismuto Ettore , Martelli Pier Luigi , Casadio Rita , Irace Gaetano
Published: February 2009

A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for β‐glycosidase from the hyperthermophilic bacterium Solfolobus sulfataricus (Sβgly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of (Sβgly) results from a bimodal distribution of fluorescence lifetimes with a short‐lived component centered at 2.5 ns and a long‐lived one at 7.4 ns Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71–79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern–Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sβgly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sβlgy evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side‐chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results.
The oxidation produced by hydrogen peroxide on Ca‐ATP‐G‐actin
Authors: Milzani Aldo , Rossi Ranieri , Simplicio Paolo Di , Giustarini Daniela , Colombo Roberto , Dalledonne Isabella
Published: February 2009

We report here that in vitro exposure of monomeric actin to hydrogen peroxide leads to a conversion of 6 of the 16 methionine residues to methionine sulfoxide residues. Although the initial effect of H2O2 on actin is the oxidation of Cys374, we have found that Met44, Met47, Met176, Met190, Met269, and Met355 are the other sites of the oxidative modification. Met44 and Met47 are the methionyl sites first oxidized. The methionine residues that are oxidized are not simply related to their accessibility to the external medium and are found in all four subdomains of actin. The conformations of subdomain 1, a region critical for the functional binding of different actin‐binding proteins, and subdomain 2, which plays important roles in the polymerization process and stabilization of the actin filament, are changed upon oxidation. The conformational changes are deduced from the increased exposure of hydrophobic residues, which correlates with methionine sulfoxide formation, from the perturbations in tryptophan fluorescence, and from the decreased susceptibility to limited proteolysis of oxidized actin.
Polypeptide stimulators of the Ms‐Lon protease
Authors: Rudyak Stanislav G. , Shrader Thomas E.
Published: February 2009

Both the peptidase activity against small fluorescent peptide substrates and the ATPase activity of Lon (La) proteases are stimulated by unstructured proteins such as α‐casein. This stimulation reveals the simultaneous interaction of Lon with two proteolytic substrates—α‐casein and the peptide substrate. To understand the cellular function of this stimulation, it is important to determine the physical properties of Lon stimulators. The abilities of compositionally simple random copolymers of amino acids (rcAAs) to stimulate the peptidase and ATPase activities of the Lon protease from Mycobacterium smegmatis (Ms‐Lon) and its N‐terminal truncation mutant (N‐E226) were determined. We report that cationic but not anionic rcAAs stimulated Ms‐Lon's peptidase activity but were themselves poor substrates for the enzyme. Peptidase stimulation by rcAAs correlated approximately with the degree of hydrophobicity of these polypeptides and reached levels >10‐fold higher than observed previously for Ms‐Lon stimulators such as α‐casein. In contrast to α‐casein, which stimulates Ms‐Lon's peptidase activity by 40% and ATPase activity by 150%, rcAAs stimulated peptidase activity without concomitant stimulation of ATPase activity. Active site labeling experiments suggested that both rcAAs and ATP increased peptidase activity by increasing accessibility to the peptidase active site. Peptidase activity assays in the presence of both α‐casein and rcAAs revealed that interactions of rcAAs and α‐casein with Ms‐Lon are extremely complex and not mutually exclusive. Specifically, (1) additions of low concentrations of α‐casein (<50 μg/mL) caused a further stimulation of Ms‐Lon's rcAA‐stimulated peptidase activity; (2) additions of higher concentrations of α‐casein inhibited Ms‐Lon's rcAA‐stimulated peptidase activity; (3) additions of all concentrations of α‐casein inhibited N‐E226′s rcAA‐stimulated peptidase activity. We conclude the Ms‐Lon can interact with an rcAA, α‐casein, and a substrate peptide simultaneously, and that formation of this quaternary complex requires the N‐terminal domain of Ms‐Lon. These data support models of Ms‐Lon that include two allosteric polypeptide binding sites distinct from the catalytic peptidase site.
Internal Auditing Research: Where are we going? Editorial
Authors: Sarens Gerrit
Published: February 2009

In this editorial, I first discuss the content of the four papers contained in this special issue. Then, I discuss certain areas for future research in internal auditing, based upon the concept of internal audit function (IAF) quality. I raise the question: ‘when can we talk about an effective IAF?’ In theory, the answer should be: ‘when IAF quality has a positive impact on the quality of corporate governance’. IAF quality consists of two components that merit further investigation: (1) the characteristics of the IAF as a whole, and (2) the characteristics of the individual internal auditor. In addition, future research should investigate to what extent IAF quality is associated with internal control quality and risk management quality, two important aspects of corporate governance. Finally, I stress the importance of the risk and control culture, when studying the relationship between IAF quality and internal control quality or risk management quality.