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Biotin protein ligase catalyses the post-translational modification of biotin carboxyl carrier protein (BCCP) domains, a modification that is crucial for the function of several carboxylases. It is a two-step process that results in the covalent attachment of biotin to the ɛ-amino group of a conserved lysine of the BCCP domain of a carboxylase in an ATP-dependent manner. In Leishmania, three mitochondrial enzymes, acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, depend on biotinylation for activity. In view of the indispensable role of the biotinylating enzyme in the activation of these carboxylases, crystal structures of L. major biotin protein ligase complexed with biotin and with biotinyl-5′-AMP have been solved. L. major biotin protein ligase crystallizes as a unique dimer formed by cross-handshake interactions of the hinge region of the two monomers formed by partial unfolding of the C-terminal domain. Interestingly, the substrate (BCCP domain)-binding site of each monomer is occupied by its own C-terminal domain in the dimer structure. This was observed in all of the crystals that were obtained, suggesting a closed/inactive conformation of the enzyme. Size-exclusion chromatography studies carried out using high protein concentrations (0.5 mM) suggest the formation of a concentration-dependent dimer that exists in equilibrium with the monomer.

Structure, stability and dynamics properties of horse cytochrome c (cyt c) and its genetically engineered M80G mutant have been investigated. The nature of the Met80 axial ligation to heme iron is believed to be the major determinant of the oxidation-reduction reactions inside and outside the cell of a particular cytochrome. This ligation has played an important role in the studies of protein structure, stability and protein folding/unfolding. To understand this ligation better, Met80 of horse cyt c has been mutated to Gly that is unable to bind to the heme iron. We have examined the effect of the M80G mutation on the structure and stability of the WT (wild type) protein by using absorbance spectroscopy, far-UV, near-UV and Soret circular dichroism, fluorescence spectroscopy and differential scanning calorimetry. We have observed that mutation caused a partial loss of secondary and tertiary structure with slightly increased overall stability of the protein. We have also measured the dynamic behavior of WT cyt c and its M80G mutant in the oxidized form (Fe3+) using the essential dynamics (ED) method. A 400 ns MD simulations were run for WT cyt c and its mutant M80G in water using GROMOS96 force field. MD results revealed that the stability and flexibility increased in mutant M80G (Fe…S (Met80) bond removed). Essential dynamics analysis revealed that the first five eigenvectors were mainly involved in overall motions of WT cyt c and its M80G mutant but the amplitude of concerted motions decreased in M80G mutant relative to WT cyt c. Communicated by Ramaswamy H. Sarma

Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25 °C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25 °C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments.

Methylcrotonyl-CoA carboxylase (MCCC) is a biotin dependent enzyme, that plays a crucial role in leucine metabolism. The enzyme comprises a biotin carboxylase (BC), a carboxyltransferase (CT), and a biotin carboxyl carrier protein (BCCP) domain. MCCC is synthesized as an apo-protein, and is posttranslationally modified at a lysine residue, conserved in the biotin carboxyl carrier protein (BCCP) domain. In order to understand the structure, function and interactions of L. major MCCC, we have expressed and characterized its domains. Here we report the complete chemical shift assignments of MCCC BCCP domain of L. major. Furthermore, we have used the assignments to generate a model of the same, using CS-Rosetta. We have also followed its chemical shift perturbations upon biotin modification. Changes were observed at the lysine 51 amide, that undergoes biotin modification, and a few others present in its immediate neighborhood.

Glycine cleavage complex H protein (GcvH) is one of the four components that form the glycine cleavage complex (GCS), essential for the synthesis of C1 (one-carbon units) for cell metabolism, by the oxidative cleavage of glycine. The activity of this complex is induced in the presence of exogenous glycine, and is repressed by purines. GCS, in cooperation with GCA (serine hydroxymethyltransferase) regulates the endogenous levels of glycine and C1 units in the cell. GcvH, the lipoamide containing component of the complex, plays an indispensable role in this reaction, as its prosthetic group shuttles between the active site of the three other components of the GCS complex sequentially. In environments rich in exogenous lipoic acid, GcvH is converted to lipoyl-GcvH by Lipoate protein ligase (LplA), by the salvage pathway. When exogenous lipoic acid is deficient, it is post-translationally modified to lipoyl-GcvH by the consecutive action of two enzymes, (a) Lipoate protein ligase B (LipB) and (b) Lipoyl synthase (LipA). Although, the crystal structure has been determined for Escherichia coli GcvH, no information exists for its interaction with LipB or LipA. Therefore, we plan to study its interactions with the aforementioned enzymes. As a first step, we have carried out the complete backbone chemical shift assignments of the E. coli glycine cleavage complex H protein in its apo-form, as well as its C8- intermediate.

Almost all proteins fold via a number of partially structured intermediates such as molten globule (MG) and pre-molten globule states. Understanding the structure of these intermediates at atomic level is often a challenge, as these states are observed under extreme conditions of pH, temperature, and chemical denaturants. Furthermore, several other processes such as chemical modification, site-directed mutagenesis (or point mutation), and cleavage of covalent bond of natural proteins often lead to MG like partially unfolded conformation. However, the dynamic nature of proteins in these states makes them unsuitable for most structure determination at atomic level. Intermediate states studied so far have been characterized mostly by circular dichroism, fluorescence, viscosity, dynamic light scattering measurements, dye binding, infrared techniques, molecular dynamics simulations, etc. There is a limited amount of structural data available on these intermediate states by nuclear magnetic resonance (NMR) and hence there is a need to characterize these states at the molecular level. In this review, we present characterization of equilibrium intermediates by biophysical techniques with special reference to NMR.

Proteins exist as conformational ensembles composed of multiple interchanging substates separated by kinetic barriers. Interconverting conformations are often difficult to probe, owing to their sparse population and transient nature. Here, we report the identification and characterization of a subset of conformations in ubiquitin that participate in microsecond-to-millisecond motions in the amides of Ile23, Asn25, and Thr55. A novel side chain to the backbone hydrogen bond that regulates these motions has also been identified. Combining our NMR studies with the available X-ray data, we have unearthed the physical process underlying slow motions-the interconversion of a type I into a type II β-turn flip at residues Glu51 through Arg54. Interestingly, the dominant conformer of wild-type ubiquitin observed in solution near neutral pH is only represented by about 22% of the crystal structures. The conformers generated as a result of the dynamics of the hydrogen bond appear to be correlated to ligand recognition by ubiquitin.

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans -hydrogen bond scalar couplings ( 3h J NC′ ) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of 3h J NC′ couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the αB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.

Previous studies of ubiquitin disclosed numerous charge–charge interactions on the protein's surface. To investigate how neighboring residues influence the strength of these interactions, double-mutant cycles are combined with pKa determinations by 2D NMR. More specifically, the environment around the Asp21–Lys29 ion pair has been altered through mutations at position 25, which is an asparagine in mammalian ubiquitin and a positively-charged residue in many other ubiquitin-like proteins. The pKa value of Asp21 decreases by 0.4 to 0.7 pH unit when Asn25 is substituted with a positively charged residue, suggesting a new and favorable ion pair interaction between positions 21 and 25. However, analysis of double mutants reveals that the favorable interaction between Asp21 and Lys29 is weakened when position 25 is a positively charged residue. Interestingly, while the pKa value of His25 in the N25H variant agrees with model compound values, additional mutants reveal that this agreement is fortuitous, resulting from a balance of favorable and unfavorable interactions; similar results were observed previously for Glu34 in ubiquitin and His8 in staphylococcal nuclease. Ionizable groups may thus have pKa values similar to model compound values and yet still be involved in significant interactions with other protein groups. One surprising result of introducing positively charged residues at position 25 is a new interaction between Lys29 and Glu18, an interaction not present in wild-type ubiquitin. This unanticipated result illustrates a key advantage of using NMR to determine pKa values for many residues simultaneously in the variant proteins. Overall, the strength of an interaction between two residues at the surface of ubiquitin is sensitive to the identity of neighboring residues. The results also demonstrate that relatively conservative and common point mutations such as substitutions of polar with charged residues and vice versa can have effects on interactions beyond the site of mutation per se.

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C(4)), -octanoyl (C(8)), -decanoyl (C(10)), -dodecanoyl (C(12)) and -tetradecanoyl (C(14))-ACPs display marked changes in backbone HN, C(alpha) and C(beta) chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.

Acyl carrier protein (ACP) is a small acidic protein, an important cofactor involved in fatty acid biosynthesis. Its main function is to protect the growing acyl chain from the hydrophilic environment during fatty acid biosynthesis and simultaneously, present it to the active site of fatty acid pathway enzymes, liable for its elongation. The ACP molecule is expressed as apo-ACP (inactive) and is post-transitionally modified to the holo form (active) by the enzyme holo ACP synthase (ACPS). Here we report the complete backbone and side chain chemical shift assignments of the holo-ACP molecule of Leishmania major.

Two highly stable cysteine proteases, ervatamin B (ERV-B) and ervatamin C (ERV-C), purified from the latex of the medicinal plant E. coronaria have been crystallized at room temperature. Crystals of ERV-B and ERV-C diffract to 2.5 and 2.6 A, respectively. The space group is P212121 for the crystals of both proteases with unit-cell parameters a = 47.5, b = 58.8 and c = 68.8 A, and a = 43.8, b = 82.6 and c = 133.1 A, respectively. A self-rotation function for ERV-C indicates a twofold non-crystallographic symmetry relating the two molecules in the asymmetric unit.

A new fluorescent amino acid provides opportunities for a better understanding of electrostatics and dynamics in proteins and their role in protein function.