Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D₂O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d₁₀. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.     

Selective editing of Val and Leu methyl groups in high molecular weight protein NMR

The development of methyl-TROSY approaches and specific (13)C-(1)H labeling of Ile, Leu and Val methyl groups in highly deuterated proteins has made it possible to study high molecular weight proteins, either alone or in complexes, using solution nuclear magnetic resonance (NMR) spectroscopy. Here we present 2-dimensional (2D) and 3-dimensional (3D) NMR experiments designed to achieve complete separation of the methyl resonances of Val and Leu, labeled using the same precursor, α-ketoisovalerate or acetolactate. The 2D experiment can further select the methyl resonances of Val or Leu based on the C(α) or C(β) chemical shift values of Val or Leu, respectively. In the 3D spectrum, the methyl cross peaks of Val and Leu residues have opposite signs; thus, not only can the residue types be easily distinguished, but the methyl pairs from the same residue can also be identified. The feasibility of this approach, implemented in both 2D and 3D experiments, has been demonstrated on an 82 kDa protein, malate synthase G. The methods developed in this study will reduce resonance overlaps and also facilitate structure-guided resonance assignments.     

A NMR experiment for simultaneous correlations of valine and leucine/isoleucine methyls with carbonyl chemical shifts in proteins

A methyl-detected ‘out-and-back’ NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts, SIM-HMCM(CGCBCA)CO, is described. The developed pulse-scheme serves the purpose of convenience in recording a single data set for all Ile^δ1, Leu^δ and Val^γ (ILV) methyl positions instead of acquiring two separate spectra selective for valine or leucine/isoleucine residues. The SIM-HMCM(CGCBCA)CO experiment can be used for ILV methyl assignments in moderately sized protein systems (up to ~100 kDa) where the backbone chemical shifts of ¹³C^α, ¹³C_β and ¹³CO are known from prior NMR studies and where some losses in sensitivity can be tolerated for the sake of an overall reduction in NMR acquisition time.     

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.     

The dissimilation of leucine, isoleucine and valine to volatile fatty acids by adult Fasciola hepatica

The dissimilation of leucine, isoleucine and valine to volatile fatty acids was determined in Fasciola hepatica and the degradation of (U−¹⁴C) branched amino acids to the volatile fatty acids end products demonstrated. F. hepatica was found to metabolize leucine, isoleucine and valine to isovaleric, 2-methylbutyric and isobutyric acid respectively. The rate of formation of isobutyrate, isovalerate and 2-methylbutyrate was found to be positively related to the rate of propionic acid production with air or nitrogen as the gas phase. However, under 95% O₂/5% CO₂ the formation of the branched chain acids was independent of propionic acid production. The production of isobutyrate, isovalerate and 2-methylbutyrate caused a simultaneous reduction in the rate of acetate formation. The role of propionate formation in regulating metabolism is discussed.     

13C-detected HN(CA)C and HMCMC experiments using a single methyl-reprotonated sample for unambiguous methyl resonance assignment

Methyl groups provide an important source of structural and dynamic information in NMR studies of proteins and their complexes. For this purpose sequence-specific assignments of methyl 1H and 13C resonances are required. In this paper we propose the use of 13C-detected 3D HN(CA)C and HMCMC experiments for assignment of methyl 1H and 13C resonances using a single selectively methyl protonated, perdeuterated and 13C/15N-labeled sample. The high resolution afforded in the 13C directly-detected dimension allows one to rapidly and unambiguously establish correlations between backbone HN strips from the 3D HN(CA)C spectrum and methyl group HmCm strips from the HMCMC spectrum by aligning all possible side-chain carbon chemical shifts and their multiplet splitting patterns. The applicability of these experiments for the assignment of methyl 1H and 13C resonances is demonstrated using the 18.6 kDa B domain of the Escherichia coli mannose transporter (IIBMannose).     

Localization of major, high molecular weight proteins in Bacteroides forsythus

Bacteroides forsythus produces species-specific major proteins with high molecular weights of 270 and 230-kDa (270K and 230K). A specific antibody raised against 270K was used for Western blot analysis and immunoelectron microscopy. Western blot analysis showed that the 270K and 230K proteins were immunologically similar. Immunogold labeling of ultrathin-sectioned bacterial cells and biochemical fractionation revealed that these proteins were localized at the outermost cell surface, not in the cytoplasm. These results suggest that major proteins ubiquitous to this species may form the S-layer.     

Susceptibility behaviour and specific heat anomaly in single crystals of alanine and valine

Magnetization of single crystals ofD-,L-alanine andD-valine were measured as a function of temperature using the SQUID magnetometer. An obvious lambda transition at 270 ± 1 K was shown in the specific heat measurement of alanine and valine enantiomers by differential scanning calorimetry (DSC). Magnetic transition temperature seems coincident with that of lambda transition. Temperature dependence of the X-ray powder diffraction forD-valine showed no crystal lattice change under the temperature cooling down from 293 K to 123 K. We propose that the difference of theX T curve between theD-alanine andL-alanine is attributable to the variation of intramolecular geometry of chirality density in the presence of an external magnetic field. The chirality characteristics of alanine and valine enantiomers by the specific heat and susceptibility behaviour are discussed.     

Development of cancer vaccines using autologous and recombinant high molecular weight stress proteins

High molecular weight heat shock proteins (HSPs), hsp110 and grp170, derived from cancer cells have been previously shown to elicit tumor-specific immunity. This phenomenon is attributed to the antigenic peptides associated with the HSPs. Based on the unique chaperoning properties of these HSPs, a new vaccination strategy has been recently developed to elicit antigen-specific antitumor immunity. This approach utilizes tumor-associated antigens naturally complexed to these highly efficient molecular chaperones under heat shock conditions. This chapter focuses on the methodologies of these two vaccine strategies: I. purification of hsp110 and grp170 from tumor tissue or cell lines; II. generation and characterization of in vitro HSP-antigen complexes by heat shock using recombinant HSPs derived from a baculovirus protein expression system.     

Clusters of isoleucine,leucine, and valine side chains define cores of stability in high‐energy states of globular proteins: Sequence determinants of structure and stability

Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high‐energy states that populate their folding free‐energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high‐energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high‐energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates.