The molecular basis for genetic polymorphism of human deoxyribonuclease II (DNase II): a single nucleotide substitution in the promoter region of human DNase II changes the promoter activity

We report that, contrary to common belief, polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein are functionally displayed on the phage surface. In a phagemid display system, carboxy-terminal fusion through optimized linker sequences resulted in display levels comparable to those achieved with conventional amino-terminal fusions. These findings are of considerable importance to phage display technology because they enable investigations not suited to amino-terminal display, including the study of protein–protein interactions requiring free carboxy-termini, functional cDNA cloning efforts, and the display of intracellular proteins.     

Perturbing probes and the study of lipid-protein bilayer membranes

Recent experimental and theoretical developments concerning perturbing probes are outlined. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene and nitroxide electron paramagnetic resonance spin labels attached to lipid hydrocarbon chains are taken as the most widely used examples of such probes. The reliability of these probes as indicators of the statics and dynamics of unlabelled lipid hydrocarbon chains is discussed, and the use of such probes in giving information about protein size, protein oligomerization and protein lateral distribution is outlined. Examples are given of studies to determine protein packing in lipid bilayers membranes.     

Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display

A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency. Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat. The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation. Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix. In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes. Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display. Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display. The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display. The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions. These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology.     

One membrane protein,two structures and six environments: a comparative molecular dynamics simulation study of the bacterial outer membrane protein PagP

PagP is a bacterial outer membrane protein consisting of an 8 stranded transmembrane β-barrel and an N-terminal α-helix. It is an enzyme which catalyses transfer of a palmitoyl chain from a phospholipid to lipid A. Molecular dynamics simulations have been used to compare the dynamic behaviour in simulations starting from two different structures (X-ray vs. NMR) and in six different environments (detergent micelles formed by dodecyl phosphocholine and by octyl glucoside, vs. four species of phospholipid bilayer). Analysis of interactions between the protein and its environment reveals the role played by the N-terminal α-helix, which interacts with the lipid headgroups to lock the PagP molecule into the bilayer. The PagP β-barrel adopts a tilted orientation in lipid bilayers, facilitating access of lipid tails into the mouth of the central binding pocket. In simulations starting from the X-ray structure in lipid bilayer, the L1 and L2 loops move towards one another, leading to the formation of a putative active site by residues H33, D76 and S77 coming closer together.     

The β5-Loop and Lid Domain Contribute to the Substrate Specificity of Pancreatic Lipase-related Protein 2 (PNLIPRP2)

Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults, whereas a homolog, pancreatic lipase-related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar, yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIPRP2 cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified, and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the surface loops tested, the lid domain and the β5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or β5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP β5-loop and decreased by the PNLIP lid domain. The source of the β9-loop had minimal effect on activity. We conclude that the lid domain and β5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2.     

Completion of the Vimentin Rod Domain Structure Using Experimental Restraints: A New Tool for Exploring Intermediate Filament Assembly and Mutations

1. Download : Download high-res image (234KB)
2. Download : Download full-size image

     

Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA)

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp⁶⁷–Glu⁷¹–Asp⁸⁰ inactivation triad within the channel structure and its bearing on the selectivity filter.     

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.     

A minimized M13 coat protein defines the requirements for assembly into the bacteriophage particle

The M13 filamentous bacteriophage coat is a symmetric array of several thousand alpha-helical major coat proteins (P8) that surround the DNA core. P8 molecules initially reside in the host membrane and subsequently transition into their role as coat proteins during the phage assembly process. A comprehensive mutational analysis of the 50-residue P8 sequence revealed that only a small subset of the side-chains were necessary for efficient incorporation into a wild-type (wt) coat. In the three-dimensional structure of P8, these side-chains cluster into three functional epitopes: a hydrophobic epitope located near the N terminus and two epitopes (one hydrophobic and the other basic) located near the C terminus on opposite faces of the helix. The results support a model for assembly in which the incorporation of P8 is mediated by intermolecular interactions involving these functional epitopes. In this model, the N-terminal hydrophobic epitope docks with P8 molecules already assembled into the phage particle in the periplasm, and the basic epitope interacts with the acidic DNA backbone in the cytoplasm. These interactions could facilitate the transition of P8 from the membrane into the assembling phage, and the incorporation of a single P8 would be completed by the docking of additional P8 molecules with the second hydrophobic epitope at the C terminus. We constructed a minimized P8 that contained only nine non-Ala side-chains yet retained all three functional epitopes. The minimized P8 assembled into the wt coat almost as efficiently as wt P8, thus defining the minimum requirements for protein incorporation into the filamentous phage coat. The results suggest possible mechanisms of natural viral evolution and establish guidelines for the artificial evolution of improved coat proteins for phage display technology.     

Decrease in 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) EPR signal in ozone-treated erythrocyte membranes

In ozone-treated erythrocyte membrane suspension a slow decrease occurs in the EPR signal of 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). Because of the absence of such a phenomenon in control membranes and ozonized buffer, this effect must be caused by reaction of nitroxide radicals with products of ozone reactions with membrane components. To find out which components are responsible for the decrease in EPR signal we studied this effect in simple model systems. The same phenomenon was observed both in lipid and protein systems treated by ozone. For unsaturated fatty acids, the correlation between the rate of decrease in EPR signal and the number of double bonds in the lipid molecule was very strong. This suggests that the observed decrease in the nitroxide radical TEMPO EPR signal in ozone-treated erythrocyte membranes is a complex process, but probably the most important reaction is recombination of nitroxide radicals with organic free radicals produced both in the process of lipid peroxidation and ozonolysis of double bonds.     

Mutations that modify or exclude binding of the QA ubiquinone and carotenoid in the reaction center from Rhodobacter sphaeroides

Three single-site mutations have been introduced at positions close to the Q_A ubiquinone in the reaction centre from Rhodobacter sphaeroides. Two of these mutations, Ala M260 to Trp and Ala M248 to Trp, result in a reaction centre that does not support photosynthetic growth of the bacterium, and in which electron transfer to the Q_A ubiquinone is abolished. In the reaction centre with an Ala to Trp mutation at the M260 residue, electron transfer from the primary donor to the acceptor bacteriopheophytin is not affected by the mutation, but electron transfer from the acceptor bacteriopheophytin to Q_A is not observed. The most likely basis for these effects is that the mutation produces a structural change that excludes binding of the Q_A ubiquinone. A third mutation, Leu M215 to Trp, produces a reaction centre that has an impaired capacity for supporting photosynthetic growth. The mutation changes the nature of ubiquinone binding at the Q_A site, and renders the site sensitive to quinone site inhibitors such as o- phenanthroline. Adopting a similar approach, in which a small residue located close to a cofactor is changed to a more bulky residue, we show that the reaction centre can be rendered carotenoid-less by the mutation Gly M71 to Leu.     

A simple quantitative method for the determination of 3-fluorotyrosine substitution in proteins

A rapid quantitative method is described for determining 3-fluorotyrosine incorporation into proteins. Derivatives of tyrosine and 3-fluorotyrosine with o-phthalaldehyde are well separated from one another by a reverse-phase high-performance liquid chromatography system used for routine analyses of o-phthalaldehyde-amino acid derivatives. Since both amino acids are well resolved from all other derivatized amino acids, the method is useful for amino acid analyses of proteins. Determination of the fluorotyrosine content of proteins by this method involves a single separation step, is reproducible, and requires no corrections for stability or yield. Further, the o-phthalaldehyde derivatives of 5-fluorotryptophan, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine can also be resolved. The method may be generally applicable to fluorinated aromatic amino acid-labeled proteins that are studied structurally and dynamically by nuclear magnetic resonance.