Single Gly Research Articles

Inner residues of macrothiolactone in autoinducer peptides I/IV circumvent spontaneous S-to-O acyl transfer to the upstream serine residue.

Autoinducing peptides I and IV (AIP-I/IV) are naturally occurring cyclic thiodepsipeptides (CTPs) bearing a Ser–Thr–Cys–Asp/Tyr (STC[D/Y]) tetrapeptide motif, where the Cys thiol (HSC) in the side-chain is linked to the Met C-terminal carboxylic acid (MCOOH) to form 5-residue macrothiolactones,−SC(D/Y)FIMCO−. We have recently reported that CTPs containing SX1CX2 motifs spontaneously undergo macrolactonization to yield cyclic depsipeptides (CDPs) by an unprecedented rapid S-to-O acyl transfer to the upstream Ser hydroxyl group. Interestingly, even though the STC[D/Y] motif in AIP-I/IV is a member of the SX1CX2 motif family, it maintains the CTP form. This suggests that AIP-I/IV have a structural or chemical motive for avoiding such an S-to-O acyl transfer, thus retaining the CTP form intact. Here we have used genetic code reprogramming to ribosomally synthesize various AIP-I analogs and studied what the determinant is to control the formation of CTP vs. CDP products. The study revealed that a Gly substitution of the inner Asp/Tyr or Met residues in the thiolactone drastically alters the resistance to the promotion of the S-to-O acyl transfer, giving the corresponding CDP product. This suggests that the steric hindrances originating from the α-substituted sidechain in these two amino acids in the AIP-I/IV thiolactone likely play a critical role in controlling the resistance against macrolactone rearrangement to the upstream Ser residue.

Modulating the Stiffness of the Myosin VI Single α-Helical Domain

Highly charged, single α-helical (SAH) domains contain a high percentage of Arg, Lys, and Glu residues. Their dynamic salt bridge pairing creates the exceptional stiffness of these helical rods, with a persistence length of more than 200 Å for the myosin VI SAH domain. With the aim of modulating the stiffness of the helical structure, we investigated the effect, using NMR spectroscopy, of substituting key charged Arg, Lys, Glu, and Asp residues by Gly or His. Results indicate that such mutations result in the transient breaking of the helix at the site of mutation but with noticeable impact on amide hydrogen exchange rates extending as far as ±2 helical turns, pointing to a substantial degree of cooperativity in SAH stability. Whereas a single Gly substitution caused transient breaks ∼20% of the time, two consecutive Gly substitutions break the helix ∼65% of the time. NMR relaxation measurements indicate that the exchange rate between an intact and a broken helix is fast (>300,000 s−1) and that for the wild-type sequence, the finite persistence length is dominated by thermal fluctuations of backbone torsion angles and H-bond lengths, not by transient helix breaking. The double mutation D27H/E28H causes a pH-dependent fraction of helix disruption, in which the helix breakage increases from 26% at pH 7.5 to 53% at pH 5.5. The ability to modulate helical integrity by pH may enable incorporation of externally tunable dynamic components in the design of molecular machines.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Detection of target collagen peptides with single amino acid mutation using two fluorescent peptide probes.

Collagen with a single amino acid substitution is the main cause of a plethora of heritable disorders such as Osteogenesis Imperfecta and Ehlers-Danlos syndrome. Though significant advances have been achieved in the development of protein assays, it remains very challenging to distinguish a protein with a single amino acid mutation from the wild-type protein. A novel fluorescent self-quenching assay has been constructed to detect target collagen peptides with a single amino acid mutation using two probe peptides. The hybridization of the probe peptide and the natural target collagen peptide results in a complete heterotrimer and strong fluorescence, whereas the mixture of the probe peptide and the mutation collagen sequences leads to a partial homotrimer and pronounced fluorescence self-quenching. The extent of fluorescence quenching is dependent on the identity of the residue replacing Gly following the order of Ala < Ser < Arg, while the Gly-Ala mutation causes the mildest fluorescence loss. The probe peptide-based fluorescence self-quenching assay facilitates specific detection of the target collagen sequence with a single Gly mutation at the nM level. The simultaneous utilization of both probe peptides enables efficient discrimination between different mutation peptides. To our knowledge, our work may be the first report of a robust analytical assay that can identify collagen fragments with single amino acid mutation, which will greatly contribute to deciphering the molecular mechanism of Osteogenesis Imperfecta as well as developing novel diagnostic strategies.

Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.

Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9 bound to RTA (2.6 Å resolution) revealed extensive antibody contact with RTA's β-strand h (732 Å2 buried surface area; BSA), along with limited engagement with α-helix D (90 Å2) and α-helix C (138 Å2). Collectively, these contacts explain the overlap between epitope clusters I and III, as identified by competition ELISA. However, considerable binding affinity, and, consequently, toxin-neutralizing activity of A9 is mediated by an unusual CDR2 containing five consecutive Gly residues that interact with α-helix B (82 Å2), a known neutralizing hotspot on RTA. Removal of a single Gly residue from the penta-glycine stretch in CDR2 reduced A9's binding affinity by 10-fold and eliminated toxin-neutralizing activity. Computational modeling indicates that removal of a Gly from CDR2 does not perturb contact with RTA per se, but results in the loss of an intramolecular hydrogen bond network involved in stabilizing CDR2 in the unbound state. These results reveal a novel configuration of a CDR2 element involved in neutralizing ricin toxin.

Comparison of bactericidal and cytotoxic activities of trichogin analogs.

Peptaibiotics are a group of membrane active peptides of fungal origin. They typically contain α-aminoisobutyric acid (Aib; 1-letter code, U) and other non-coded residues (Toniolo and Brückner, 2009; Neumann et al., 2015; Benedett et al., 1982) [1], [2], [3] stabilizing their helical structure. Peptaibols are peptaibiotics carrying a 1, 2-aminoalcohol at the C-terminus. When a fatty acid chain (of 8–10 carbon atoms) is present at their N-terminus, they are called lipopeptaibols (Toniolo et al., 2001; Degenkolb et al., 2003) [4], [5]. We found (Tavano et al., 2015) [6] that the lipopeptaibol trichogin displays no antibacterial effects up to 64 µM, against both Gram− and Gram+ bacteria, but kills tumor and healthy human cells via a mechanism requiring both the C-terminal primary alcohol group and the N-terminal n-octanoyl moiety, with EC50s around 4–5 µM. However, the substitution of single Gly residues with Lys strongly improves anti-Gram+ activity (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion et al., 2012) [6], [7], [8]. To further characterize the activity of trichogin analogs as antibiotics and cytotoxic agents, we here manipulated the peptide helix amphipathicity by means of two different substitutions: (i) Aib to Leu (De Zotti et al., 2012) [7] or (ii) multiple Gly to Lys changes (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion, Formaggio et al., 2012; De Zotti, Biondi, Peggion, De Poli et al., 2012) [6], [7], [8], [9]. The antibacterial activity against four commensal or opportunistic bacterial species and the cytotoxicity against a panel of 9 healthy and tumor-derived eukaryotic cell types (including erythrocytes) are reported as MIC and EC50 (MTS - [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)]-2H-tetrazolium- reduction and LDH - lactate dehydrogenase - release assay).

CD and NMR investigation of collagen peptides mimicking a pathological Gly-Ser mutation and a natural interruption in a similar highly charged sequence context.

Even a single Gly substitution in the triple helix domain of collagen leads to pathological conditions while natural interruptions are suggested to play important functional roles. Two peptides-one mimicking a pathological Gly-Ser substitution (ERSEQ) and the other one modeling a similar natural interruption sequence (DRSER)-are designed to facilitate the comparison for elucidating the molecular basis of their different biological roles. CD and NMR investigation of peptide ERSEQ indicates a reduction of the thermal stability and disruption of hydrogen bonding at the Ser mutation site, providing a structural basis of the OI disease resulting from the Gly-Ser mutation in the highly charged RGE environment. Both CD and NMR real-time folding results indicate that peptide ERSEQ displays a comparatively slower folding rate than peptide DRSER, suggesting that the Gly-Ser mutation may lead to a larger interference in folding than the natural interruption in a similar RSE context. Our studies suggest that unlike the rigid GPO environment, the abundant R(K)GE(D) motif may provide a more flexible sequence environment that better accommodates mutations as well as interruptions, while the electrostatic interactions contribute to its stability. These results shed insight into the molecular features of the highly charged motif and may aid the design of collagen biomimetic peptides containing important biological sites.

Modulation of Cytochrome P450 Activity by 18β-Glycyrrhetic Acid and its Consequence on Buspirone Pharmacokinetics in Rats.

The aim of this study was to elucidate the inhibition mechanism of 18β-glycyrrhetic acid (GLY) on cytochrome P450 (CYP) activity and in vivo pharmacokinetic consequences of single GLY dose in rats. An in vitro CYP inhibition study in rat liver microsomes (RLM) was conducted using probe substrates for CYPs. Then, an in vivo pharmacokinetics of intravenous and oral buspirone (BUS), a probe substrate for CYP3A, was studied with the concurrent administration of oral GLY in rats. In the in vitro CYP inhibition study, CYP3A was involved in the metabolism of GLY. Moreover, GLY inhibited CYP3A activity with an IC50 of 20.1 ± 10.7 μM via a mixed inhibition mechanism. In the in vivo rat pharmacokinetic study, single oral GLY dose enhanced the area under plasma concentration-time curve (AUC) of intravenous and oral BUS, but the extent of increase in AUC was only minimal (1.12-1.45 fold). These results indicate that GLY can inhibit the in vitro CYP3A-mediated drug metabolism in RLM via a mixed inhibition mechanism. However, the impact of single oral GLY dose on the pharmacokinetics of BUS in rats was limited, showing that GLY could function as merely a weak inhibitor for CYP3A-mediated drug metabolism in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

Local amino acid sequence patterns dominate the heterogeneous phenotype for the collagen connective tissue disease Osteogenesis Imperfecta resulting from Gly mutations.

Osteogenesis Imperfecta (OI), a hereditary connective tissue disease in collagen that arises from a single Gly → X mutation in the collagen chain, varies widely in phenotype from perinatal lethal to mild. It is unclear why there is such a large variation in the severity of the disease considering the repeating (Gly-X-Y)n sequence and the uniform rod-like structure of collagen. We systematically evaluate the effect of local (Gly-X-Y)n sequence around the mutation site on OI phenotype using integrated bio-statistical approaches, including odds ratio analysis and decision tree modeling. We show that different Gly → X mutations have different local sequence patterns that are correlated with lethal and nonlethal phenotypes providing a mechanism for understanding the sensitivity of local context in defining lethal and non-lethal OI. A number of important trends about which factors are related to OI phenotypes are revealed by the bio-statistical analyses; most striking is the complementary relationship between the placement of Pro residues and small residues and their correlation to OI phenotype. When Pro is present or small flexible residues are absent nearby a mutation site, the OI case tends to be lethal; when Pro is present or small flexible residues are absent further away from the mutation site, the OI case tends to be nonlethal. The analysis also reveals the dominant role of local sequence around mutation sites in the Major Ligand Binding Regions that are primarily responsible for collagen binding to its receptors and shows that non-lethal mutations are highly predicted by local sequence considerations alone whereas lethal mutations are not as easily predicted and may be a result of more complex interactions. Understanding the sequence determinants of OI mutations will enhance genetic counseling and help establish which steps in the collagen hierarchy to target for drug therapy.

First-principles studies of peptide fibres

The electronic properties of a single Gly, Cys and Trp peptide fibres (PFs), in separated and closely spaced forms, are studied with ab initio calculations. By decreasing the distance between separated Gly and Cys PFs, respectively, the energy gap between highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of separated fibres tends to narrow down and we investigate the smallest HOMO–LUMO gap for the separated Trp PFs of 3.36 eV, without any electronic states between the gap. The band gap of closely spaced Trp and Gly PFs is predicted to be 0.26 and 3.31 eV, indicating an organic semiconductor character. Series of deep electronic bands (DEBs) in the band gap are found for the closely spaced Cys PFs, helping recombination in the system with the largest DEB gap of 2.26 eV. The electronic properties of all three fibres are mainly decided by the peptide bonds and the functional side chains. The results show that design of the side chains on the PF backbone is a promising method for the application of organic semiconductor and biological compatible devices.

Gly or Ala substitutions for Pro210Thr211Asn212 at the β8–β9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability

A comparison of the primary structures among psychrophilic, mesophilic, and thermophilic subtilases revealed that the turn between the β8 and β9 strands (β8–β9 turn, BPN′ numbering) of psychrophilic subtilases are more flexible than those of their mesophilic and thermophilic counterparts. To investigate the relationship between structure of this turn and enzyme activity as well as thermostability of mesophilic subtilisin Carlsberg (sC), we analyzed 6 mutants of sC with a single, double, or triple Gly or Ala substitutions for Pro210Thr211Asn212 at the β8–β9 turn. Among the single Gly substitutions, the P210G substitution most significantly (1.5-fold) increased the specific activity on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF) substrate and 12-fold decreased the thermostability. All mutants tested showed the increased kcat for the AAPF substrate and reduced thermostability compared with the wild-type sC. The kcat values of the P210G, P210G/T211G, and P210G/T211G/N212G mutants were 1.5-, 1.7-, and 1.8-fold higher than that of the wild-type sC. There were significant positive correlations between kcat and thermal inactivation rates as well as kcat and Km of the wild-type and mutants. These results demonstrate that the structure of β8–β9 turn, despite its distance from the active site, has significant effects on the catalytic rate and thermostability of sC through a global network of intramolecular interactions and suggest that the lack of flexibility of this turn stabilizes the wild-type sC against thermal inactivation in compensation for some loss of catalytic activity.

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.

Interruption of a 310‐helix by a single Gly residue in a poly‐Aib motif: A crystallographic study

The structural influence of a single Gly residue inserted into an Aib(16) homooligomer was studied in the solid state by X-ray crystallography. The peptides N(3)Aib(8)GlyAib(8)PheNH(2) (1) and CbzPheAib(8)GlyAib(8) (2) were found to adopt well-defined helical structures, which are broadly 3(10) helical. Indeed, 2 is the longest crystallographic 3(10) helix thus far reported. However, in the region of the central Gly residue, a loosening of the 3(10) structure is observed in both peptides, with 1 clearly showing local adoption of an α-helical structure in the region of residues 7-9.

Development of crosslink‐type tag‐probe pairs for fluorescent imaging of proteins

Useful methods of protein labeling via functional peptide tags have been developed in the field of proteome and chemical biology. New tag-probe pairs based on leucine zipper peptides for labeling target proteins are described. These consist of an α-helical probe peptide with an environmental-sensitive fluorescent dye and two antiparallel α-helical tag peptides, and may be crosslinked, from the Cys residue of the tag peptide to the N(α)-chloroacetyl group of the probe peptide. Binding of the probe peptide to the tag peptides results in movement of the fluorophore from a hydrophilic to a hydrophobic environment inside the leucine zipper structure, causing a dramatic fluorescent change, mediated by the binding of the two peptides. As a spacer between the N(α)-chloroacetyl group and the original probe sequence, a single Gly residue was the most suitable among 0-2 Gly residues. Crosslinking leads to superior fluorescence response, binding affinity, and chemical stability. These ZIP tag-probe pairs are useful for labeling and fluorescent imaging of proteins.

Sus1, Cdc31, and the Sac3 CID Region Form a Conserved Interaction Platform that Promotes Nuclear Pore Association and mRNA Export

SummaryThe yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)—“gene gating”—that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3CID forms a long, gently undulating α helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3CID provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.

Hb Dothan [β25/26 (B7/B8)/(-GTG/-GLY)/Gly+Glu→Glu]; A Novel Mechanism Leading to a M-Hemoglobin.

Hereditary methemoglobinemia is a relatively rare disorder usually manifesting with cyanosis at birth. The more common form results from the deficiency of the enzyme, NADH-Cytochrome b5 reductase (methemoglobin reductase, diaphorase) and displays an autosomal recessive inheritance pattern. Less common are the so-called M-hemoglobins with an autosomal dominant pattern, which result from amino acid substitutions in the heme binding pocket of α, β, or less commonly γ-globin chains. The majority of the M-hemoglobin (Hb) variants occur from substitutions in the E or F-helices, which constitute the heme binding pocket, most commonly from amino acid substitutions involving the conserved proximal (F8) or distal (E11) histidine residues. Here we report a new Hb variant due to a three nucleotide deletion (-GTG between codons 25 and 26 of the β globin gene causing a single amino acid (-Gly) deletion in the B helix (B7/B8) of the β-globin chain that leads to methemoglobinemia with a novel mechanism. The propositus is a 9 month old Caucasian boy from Dothan AL who was found to have a low O2 saturation prior to an ENT procedure. He was referred to cardiology at Children's Health System, Birmingham, AL to rule out a congenital heart disease. A low O2 saturation (85–86%) was confirmed. Cardiac catheterization excluded the structural abnormality of the heart. Cooximetry showed a normal PaO2 but confirmed a low O2 saturation. Methemoglobin level was 20%, while methemoglobin reductase activity was in the low–normal range but when repeated was found to be normal. His growth and development have been normal. On alkaline electrophoresis an abnormal hemoglobin band was observed. The patient's blood was sent to the Hemoglobinopathy Laboratory of the Sickle Cell Center at MCG, Augusta, GA for definite identification of the variant. CBC revealed a RBC of 4.3 M/mm3, HGB 13.6 g/dL, HCT 40.8 %, MCV 95.2 fl, MCH 31.8 pg MCHC 33.4 g/dL, Retics 4.4 %. Isoelectrofocusing (IEF) on agarose showed the presence of an abnormal Hb with approximately the same isoelectric point (pI) as Hb F. Quantitation of Hb components by Cation Exchange HPLC revealed 62.7% Hb A, 27.9% Hb X, 3.0% Hb A2, and 6.4% Hb F. By globin chain analyses with reversed phase HPLC, βχ was detected as 37.6% of the total beta chains. Isopropanol stability test gave strongly positive results. P50 was found to be 24.8 mm Hg in the patient and 26.4 in the control (slightly increased oxygen affinity). Peptide analysis was done using mass spectrometry (Alphalyse, Palo Alto, CA) where tryptic digests of purified Hb X (95.0% enriched) and normal control (97.0% Hb A) were analyzed and compared. Peptide 19–30 of helix-B fragment revealed 1314 Da mass in control, whereas peptide 19–29 (with –Gly) of helix-B fragment of Hb X gave 1257 Da mass, confirming the deletion of a Gly residue. The corresponding deletion of three nucleotides (-GTG) in the genomic DNA (codons 25–26: GTGGAG→GAG ) was demonstrated by polymerase chain reaction (PCR) amplification and direct sequencing of β-globin gene and confirmed by cDNA sequencing of β-globin mRNA. No abnormality was detected in the sequences of δ, Gγ, Aγ, α1 and α2 globin genes. The three nucleotide deletion between codons 25 and 26 (-GTG) of the β-globin gene causes a one amino acid (-Gly) deletion in the B helix (B7/B8) of the β-globin chain, however does not alter the amino acid composition of β-globin chain after the deletion point but results in a shorter (145 AA, instead of the normal 146) mutant β-globin chain. As a result close spatial contact of amino acids in tertiary structure of hemoglobin is altered completely. Most importantly, distal histidine at residue 63 of E7 helix now becomes Gly leading to methemoglobin formation. A similar variant was previously reported in a Japanese baby, Hb Higashitochigi (Fujisawa et al, Hemoglobin , 17:467, 1993) where a three nucleotide deletion in codons 24/25 also resulted in the loss of a single Gly residue with a similar outcome. These two cases differ from the known M-hemoglobins all of which result from single amino acid substitution in the E or F-helices thus altering the heme pocket. Hb Dothan and Hb Higashitochigi represent a novel mechanism for M-hemoglobin generation where an in frame deletion alters the tertiary structure of the globin chain with alterations in the structure of E-helix and loss of the distal histidine residue.

Sequence dependence of kinetics and morphology of collagen model peptide self‐assembly into higher order structures

The process of self-assembly of the triple-helical peptide (Pro-Hyp-Gly)(10) into higher order structure resembles the nucleation-growth mechanism of collagen fibril formation in many features, but the irregular morphology of the self-assembled peptide contrasts with the ordered fibers and networks formed by collagen in vivo. The amino acid sequence in the central region of the (Pro-Hyp-Gly)(10) peptide was varied and found to affect the kinetics of self-assembly and nature of the higher order structure formed. Single amino acid changes in the central triplet produced irregular higher order structures similar to (Pro-Hyp-Gly)(10), but the rate of self-association was markedly delayed by a single change in one Pro to Ala or Leu. The introduction of a Hyp-rich hydrophobic sequence from type IV collagen resulted in a more regular suprastructure of extended fibers that sometimes showed supercoiling and branching features similar to those seen for type IV collagen in the basement membrane network. Several peptides, where central Pro-Hyp sequences were replaced by charged residues or a nine-residue hydrophobic region from type III collagen, lost the ability to self-associate under standard conditions. The inability to self-assemble likely results from loss of imino acids, and lack of an appropriate distribution of hydrophobic/electrostatic residues. The effect of replacement of a single Gly residue was also examined, as a model for collagen diseases such as osteogenesis imperfecta and Alport syndrome. Unexpectedly, the Gly to Ala replacement interfered with self-assembly of (Pro-Hyp-Gly)(10), while the peptide with a Gly to Ser substitution self-associated to form a fibrillar structure.

Immobilization of oligoglycines on aldehyde-decorated surfaces and its influence on the orientations of liquid crystals

This paper reports an investigation of the immobilization of glycine (Gly), glycyl-glycine (Gly-Gly), and glycyl-glycyl-glycine (Gly-Gly-Gly) on silica substrates (colloidal silica, silicon wafer, and glass slides) decorated with aldehyde functional groups. It is found that the density of Gly or oligoglycines immobilized on the surface can be greatly enhanced by using higher ionic strength (∼1 M) and a reaction temperature of 50 °C. High ionic strength was used to screen the electrostatic repulsion caused by the negatively charged carboxylate groups of Gly or oligoglycines, while higher temperature was needed to increase the reaction rates. It is also found that when a thin layer of nematic liquid crystal 4-pentyl-4′-cyano-biphenyl (5CB) was supported on a Gly or oligoglycine-decorated surface, the orientation of 5CB changed from planar to homeotropic (perpendicular to the surface) over a period of 24 h. Furthermore, the switching time decreased when the surface density of oligoglycines was increased, and when the length of oligoglycines was increased by one or two Gly residues. The kinetic information provides a means to distinguish Gly, Gly-Gly, and Gly-Gly-Gly that only differ by a single Gly without using fluorescence or radioactive labeling. This method also has good spatial resolution over a large area that permits the detection of multiple oligoglycines patterned on the same surface.

Two Novel Ubiquitin-fold Modifier 1 (Ufm1)-specific Proteases, UfSP1 and UfSP2

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.

Transformation of the Mechanism of Triple-helix Peptide Folding in the Absence of a C-terminal Nucleation Domain and Its Implications for Mutations in Collagen Disorders

Folding abnormalities of the triple helix have been demonstrated in collagen diseases such as osteogenesis imperfecta in which the mutation leads to the substitution of a single Gly in the (Gly-X-Y)n sequence pattern by a larger residue. Model peptides can be used to clarify the details of normal collagen folding and the consequences of the interruption of that folding by a Gly substitution. NMR and CD studies show that placement of a (GPO)4 nucleation domain at the N terminus rather than the C terminus of a native collagen sequence allows the formation of a stable triple helix but alters the folding mechanism. Although C- to N-terminal directional folding occurs when the nucleation domain is at the C terminus, there is no preferential folding direction when the nucleation domain is at the N terminus. The lack of zipper-like directional folding does not interfere with triple-helix formation, and when a Gly residue is replaced by Ser to model an osteogenesis imperfecta mutation, the peptide with the N-terminal (GPO)4 domain can still form a good triple helix N-terminal to the mutation site. These peptide studies raise the possibility that mutant collagen could fold in a C to N direction in a zipper-like manner up to the mutation site and that completion of the triple helix N-terminal to the mutation would involve an alternative mechanism.