Biomedical subjects

Robert Fairman

Publications and source records attributed to Robert Fairman.

At least 19 recordsLinked to original sources

PubMed2006

Helix propensity of highly fluorinated amino acids.

Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. To gain further insight into the effect of these highly fluorinated amino acids on helix formation exclusively, we measured the helix propensity of three highly fluorinated amino acids: (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), and (S)-pentafluorophenylalanine (Pff). We have developed a short chemoenzymatic synthesis of Hfl with extremely high enantioselectivity (>99%). To measure the helix propensity (w) of the amino acids, alanine-based peptides were synthesized, purified, and investigated by circular dichroism spectroscopy (CD). On the basis of the CD data, the helix propensity of hydrocarbon amino acids can decrease up to 24-fold (1.72 kcal.mol-1.residue-1) upon fluorination. This difference in helix propensity has previously been overlooked in estimating the magnitude of the fluoro-stabilization effect (which has been estimated to be 0.32-0.83 kcal.mol-1.residue-1 for Hfl), resulting in a gross underestimation. Therefore, the full potential of the fluoro-stabilization effect should provide even more stable proteins than the fluoro-stabilized proteins to date.

Amino Acids↗

PubMed2006

Self-assembly of peptide porphyrin complexes: toward the development of smart biomaterials.

The anionic porphyrin, meso-tetrakis(4-sulfonatophenyl)porphine, is found to tightly bind to an engineered 14-residue peptide, resulting in induced alpha-helix formation when mixed in aqueous solutions. The small porphyrin-peptide dissociation constant (2 muM) observed is related to the energetics of peptide helix formation coupled with electrostatic interactions between the anionic porphyrin and cationic residues in the coiled peptide. Analytical ultracentrifugation measurements indicate the porphyrin-peptide complexes dimerize, probably into a coiled coil, and weakly associate to form even higher order structures.

Amino Acid Sequence↗

PubMed2006

Crystal structure of a glycyl radical enzyme from Archaeoglobus fulgidus.

We have solved the crystal structure of a PFL2 from Archaeglobus fulgidus at 2.9 A resolution. Of the three previously solved enzyme structures of glycyl radical enzymes, pyruvate formate lyase (PFL), anaerobic ribonucleotide reductase and glycerol dehydratase (GD), the last one is clearly most similar to PFL2. We observed electron density in the active site of PFL2, which we modelled as glycerol. The orientation of the glycerol is different from that in GD, and changes in the active site indicate that the actual substrate of PFL2 is bigger than a glycerol molecule, but sequence and structural homology suggest that PFL2 may be a dehydratase. Crystal packing, solution X-ray scattering and ultracentrifugation experiments show that PFL2 is tetrameric, unlike other glycyl radical enzymes. A.fulgidus is a hyperthermophile and PFL2 appears to be stabilized by several factors including an increased number of ion pairs, differences in buried charges, a truncated N terminus, anchoring of loops and N terminus via salt-bridges, changes in the oligomeric interface and perhaps also the higher oligomerization state of the protein.

Acetyltransferases↗

PubMed2005

Single amino acid mutations alter the distribution of human porphobilinogen synthase quaternary structure isoforms (morpheeins).

Porphobilinogen synthase (PBGS) is an obligate oligomer that can exist in functionally distinct quaternary states of different stoichiometries, which are called morpheeins. The morpheein concept describes an ensemble of quaternary structure isoforms wherein different structures of the monomer dictate different multiplicities of the oligomer (Jaffe, E. K. (2005) Trends Biochem. Sci. 30, 490-497). Human PBGS assembles into long-lived morpheeins and has been shown to be capable of forming either a high activity octamer or a low activity hexamer (Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A., and Jaffe, E. K. (2003) Nat. Struct. Biol. 10, 757-763). All PBGS monomers contain an alphabeta-barrel domain and an N-terminal arm domain. The N-terminal arm structure varies among PBGS morpheeins, and the spatial relationship between the arm and the barrel dictates the different quaternary assemblies. We have analyzed the structures of human PBGS morpheeins for key interactions that would be predicted to affect the oligomeric assembly. Examples of individual mutations that shift assembly of human PBGS away from the native octamer are R240A and W19A. The alternate morpheeins of human PBGS variants R240A and W19A are chromatographically separable from each other and kinetically distinct; their structure and dynamics have been characterized by native gel electrophoresis, dynamic light scattering, and analytical ultracentrifugation. R240A assembles into a metastable hexamer, which can undergo a reversible conversion to the octamer in the presence of substrate. The metastable nature of the R240A hexamer supports the hypothesis that octameric and hexameric morpheeins of PBGS are very close in energy. W19A assembles into a mixture of dimers, which appear to be stable.

Amino Acid Substitution↗

PubMed2005

Calbindin D28k EF-hand ligand binding and oligomerization: four high-affinity sites--three modes of action.

Calbindin D28k, a highly conserved protein with Ca2+-sensing and Ca2+-buffering capabilities, is abundant in brain and sensory neurons. This protein contains six EF-hand subdomains, four of which bind Ca2+ with high affinity. Calbindin D28k can be reconstituted from six synthetic peptides corresponding to the six EF-hands, indicating a single-domain structure with multiple interactions between the EF-hand subdomains. In this study, we have undertaken a detailed characterization of the Ca2+-binding and oligomerization properties of each individual EF-hand peptide using CD spectroscopy and analytical ultracentrifugation. Under the conditions tested, EF2 is monomeric and does not bind Ca2+, whereas EF6, which binds Ca2+ weakly, aggregates severely. We have therefore focused this study on the high-affinity binding sites, EF-hands 1, 3, 4, and 5. Our sedimentation equilibrium data show that, in the presence of Ca2+, EF-hands 1, 3, 4, and 5 all form dimers in solution in which the distribution between the monomer, dimer, and higher order oligomers differs. The processes of Ca2+ binding and oligomerization are linked to different degrees, and three main mechanisms emerge. For EF-hands 1 and 5, the dimer binds Ca2+ more strongly than the monomer and Ca2+ binding drives dimerization. For EF-hand 4, dimer formation requires only one of the monomers to be Ca2+-bound. In this case, the Ca2+ affinity is independent of dimerization. For EF-hand 3, dimerization occurs both in the absence and presence of Ca2+, while oligomerization increases in the presence of Ca2+.

Amino Acid Sequence↗

PubMed2005

Quantitative atomic force microscopy image analysis of unusual filaments formed by the Acanthamoeba castellanii myosin II rod domain.

We describe a quantitative analysis of Acanthamoeba castellanii myosin II rod domain images collected from atomic force microscope experiments. These images reveal that the rod domain forms a novel filament structure, most likely requiring unusual head-to-tail interactions. Similar filaments are seen also in negatively stained electron microscopy images. Truncated myosins from Acanthamoeba and other model organisms have been visualized before, revealing laterally associated bipolar minifilaments. In contrast, the filament structures that we observe are dominated by axial rather than lateral polymerization. The unusually small features in this structure (1-5 nm) required the development of quantitative and statistical techniques for filament image analysis. These techniques enhance the extraction of features that hitherto have been difficult to ascertain from more qualitative imaging approaches. The heights of the filaments are observed to have a bimodal distribution consistent with the diameters of a single rod domain and a pair of close-packed rod domains. Further quantitative analysis indicates that in-plane association is limited to at most a pair of rod domains. Taken together, this implies that the filaments contain no more than four rod domains laterally associated with one another, somewhat less than that seen in bipolar minifilaments. Analysis of images of the filaments decorated with an anti-FLAG antibody reveals head-to-tail association with mean distances between the antibodies of 75 +/- 15 nm. We consider a set of molecular models to help interpret possible structures of the filaments.

Acanthamoeba castellanii↗

PubMed2005

Toward the development of peptide nanofilaments and nanoropes as smart materials.

Protein design studies using coiled coils have illustrated the potential of engineering simple peptides to self-associate into polymers and networks. Although basic aspects of self-assembly in protein systems have been demonstrated, it remains a major challenge to create materials whose large-scale structures are well determined from design of local protein-protein interactions. Here, we show the design and characterization of a helical peptide, which uses phased hydrophobic interactions to drive assembly into nanofilaments and fibrils ("nanoropes"). Using the hydrophobic effect to drive self-assembly circumvents problems of uncontrolled self-assembly seen in previous approaches that used electrostatics as a mode for self-assembly. The nanostructures designed here are characterized by biophysical methods including analytical ultracentrifugation, dynamic light scattering, and circular dichroism to measure their solution properties, and atomic force microscopy to study their behavior on surfaces. Additionally, the assembly of such structures can be predictably regulated by using various environmental factors, such as pH, salt, other molecular crowding reagents, and specifically designed "capping" peptides. This ability to regulate self-assembly is a critical feature in creating smart peptide biomaterials.

Amino Acid Sequence↗

PubMed2005

Self-assembling porphyrin-modified peptides.

[structure: see text] We report the synthesis and characterization of a novel supramolecular assembly that features long-range electronic coupling between porphyrins covalently attached to a designed peptide scaffold. The resulting construct self-assembles to form extended organized aggregates in which the porphyrins engage in exciton coupling.

Hydrogen-Ion Concentration↗

PubMed2005

Engineering a dimeric caspase-9: a re-evaluation of the induced proximity model for caspase activation.

Caspases are responsible for the execution of programmed cell death (apoptosis) and must undergo proteolytic activation, in response to apoptotic stimuli, to function. The mechanism of initiator caspase activation has been generalized by the induced proximity model, which is thought to drive dimerization-mediated activation of caspases. The initiator caspase, caspase-9, exists predominantly as a monomer in solution. To examine the induced proximity model, we engineered a constitutively dimeric caspase-9 by relieving steric hindrance at the dimer interface. Crystal structure of the engineered caspase-9 closely resembles that of the wild-type (WT) caspase-9, including all relevant structural details and the asymmetric nature of two monomers. Compared to the WT caspase-9, this engineered dimer exhibits a higher level of catalytic activity in vitro and induces more efficient cell death when expressed. However, the catalytic activity of the dimeric caspase-9 is only a small fraction of that for the Apaf-1-activated caspase-9. Furthermore, in contrast to the WT caspase-9, the activity of the dimeric caspase-9 can no longer be significantly enhanced in an Apaf-1-dependent manner. These findings suggest that dimerization of caspase-9 may be qualitatively different from its activation by Apaf-1, and in conjunction with other evidence, posit an induced conformation model for the activation of initiator caspases.

Caspase 9↗

PubMed2005

Peptides as novel smart materials.

Important challenges in biomaterials design include predicting the formation of large-scale self-assembled structures based on local atomic-level interactions and then endowing such structures with the ability to respond sensitively to environmental cues. This responsiveness is referred to as smartness. With the advent of key technological advances in imaging, peptides have recently begun to be exploited for their potential use as biomaterials, such as filaments and fibrils, hydrogels, surfactants and peptide hybrids. Peptides offer attractive features, principally because of our detailed understanding of their ability to fold into specific structures, and the rich chemistry with which their structure and function can be manipulated for environmental response.

Biocompatible Materials↗

PubMed2004

Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer.

BACKGROUND: The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus. RESULTS: The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of approximately 6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed. CONCLUSION: The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins.

Amino Acid Sequence↗

PubMed2004

Protein dissection experiments reveal key differences in the equilibrium folding of alpha-lactalbumin and the calcium binding lysozymes.

The alpha-lactalbumins and c-type lysozymes have virtually identical structure but exhibit very different folding behavior. All alpha-lactalbumins form a well populated molten globule state, while most of the lysozymes do not. alpha-Lactalbumin consists of two subdomains, and the alpha-subdomain is considerably more structured in the molten globule state than the beta-subdomain. Constructs derived from the alpha-subdomain of human alpha-lactalbumin containing the A, B, D, and 3(10) helices are known to form a molten globule state in the absence of the rest of the protein (Demarest, S. et al. (1999) J. Mol. Biol. 294, 213-221). Here we reported comparative studies of constructs derived from the same regions of canine and equine lysozymes. These proteins form two of the most stable molten globule states among all the lysozymes. A construct containing the A, B, D, and 3(10) helices of equine lysozyme is partially helical but is less structured than the corresponding human alpha-lactalbumin peptide. Addition of the C-helix leads to a construct that is still less structured and less stable than the alpha-lactalbumin construct. The corresponding construct from canine lysozyme is also less structured and less stable than the alpha-lactalbumin peptide. Thus, molten globule formation in human alpha-lactalbumin can be driven by the isolated alpha-subdomain, while more extensive interactions are required to generate a stable molten globule in the two lysozymes. The stability of the canine and equine lysozyme constructs is similar, indicating that the extraordinary stability of the canine lysozyme molten globule is not due to an unusually stable isolated alpha-subdomain.

Amino Acid Sequence↗

PubMed2004

Peptide models provide evidence for significant structure in the denatured state of a rapidly folding protein: the villin headpiece subdomain.

The villin headpiece subdomain is a cooperatively folded 36-residue, three-alpha-helix protein. The domain is one of the smallest naturally occurring sequences which has been shown to fold. Recent experimental studies have shown that it folds on the 10-micros time scale. Its small size, simple topology, and very rapid folding have made it an attractive target for computational studies of protein folding. We present temperature-dependent NMR studies that provide evidence for significant structure in the denatured state of the headpiece subdomain. A set of peptide fragments derived from the headpiece were also characterized in order to determine if there is a significant tendency to form a locally stabilized structure in the denatured state. Peptides corresponding to each of the three isolated helices and to the connection between the first and second helices were largely unstructured. A longer peptide fragment which contains the first and second helices shows considerable structure, as judged by NMR and CD. Concentration-dependent CD measurements and analytical ultracentrifugation experiments indicate that the structure is not due to self-association. NMR studies indicate that the structure is stabilized by tertiary interactions involving phenylalanines and Val 50. A peptide in which two of the three phenylalanines are changed to leucine is considerably less structured, confirming the importance of the phenylalanines. This work indicates that there is significant structure in the denatured state of this rapidly folding protein.

Amino Acid Sequence↗

PubMed2003

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.

Allosteric Site↗

PubMed2003

The SNARE motif contributes to rbet1 intracellular targeting and dynamics independently of SNARE interactions.

The endoplasmic reticulum/Golgi SNARE rbet1 cycles between the endoplasmic reticulum and Golgi and is essential for cargo transport in the secretory pathway. Although the quaternary SNARE complex containing rbet1 is known to function in membrane fusion, the structural role of rbet1 is unclear. Furthermore, the structural determinants for rbet1 targeting and its cyclical itinerary have not been investigated. We utilized protein interaction assays to demonstrate that the rbet1 SNARE motif plays a structural role similar to the carboxyl-terminal helix of SNAP-25 in the synaptic SNARE complex and demonstrated the importance to SNARE complex assembly of a conserved salt bridge between rbet1 and sec22b. We also examined the potential role of the rbet1 SNARE motif and SNARE interactions in rbet1 localization and dynamics. We found that, in contrast to what has been observed for syntaxin 5, the rbet1 SNARE motif was essential for proper targeting. To test whether SNARE interactions were important for the targeting function of the SNARE motif, we used charge repulsion mutations at the conserved salt bridge position that rendered rbet1 defective for binary, ternary, and quaternary SNARE interactions. We found that heteromeric SNARE interactions are not required at any step in rbet1 targeting or dynamics. Furthermore, the heteromeric state of the SNARE motif does not influence its interaction with the COPI coat or efficient recruitment onto transport vesicles. We conclude that protein targeting is a completely independent function of the rbet1 SNARE motif, which is capable of distinct classes of protein interactions.

Amino Acid Motifs↗

PubMed2003

Mechanism of XIAP-mediated inhibition of caspase-9.

The inhibitor of apoptosis (IAP) proteins potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 A resolution. The structure reveals that the BIR3 domain forms a heterodimer with a caspase-9 monomer. Strikingly, the surface of caspase-9 that interacts with BIR3 also mediates its homodimerization. We demonstrate that monomeric caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases.

Caspase 9↗

PubMed2002

Characterization of large peptide fragments derived from the N-terminal domain of the ribosomal protein L9: definition of the minimum folding motif and characterization of local electrostatic interactions.

A set of peptides derived from the N-terminal domain of the ribosomal protein L9 (NTL9) have been characterized in an effort to define the minimum unit of this domain required to fold and to provide model peptides for the analysis of electrostatic interactions in the unfolded state. NTL9 is a 56-residue alpha-beta protein with a beta1-loop-beta2-alpha1-beta3-alpha2 topology. The beta-sheet together with the first helix comprise a simple example of a common supersecondary motif called the split beta-alpha-beta fold. Peptides corresponding to the beta1-loop-beta2 unit are unstructured even when constrained by an introduced disulfide. The pK(a)s of Asp-8 and Glu-17 in these peptides are slightly lower than the values found for shorter peptides but are considerably higher than the values in NTL9. A 34-residue peptide, which represents the beta1-loop-beta2-alpha1 portion of NTL9, is also unstructured. In contrast, a 39-residue peptide corresponding to the entire split beta-alpha-beta motif is folded and monomeric as judged by near- and far-UV CD, two-dimensional NMR, ANS binding experiments, pK(a) measurements, and analytical ultracentrifugation. The fold is very similar to the structure of this region in the intact protein. Thermal and urea unfolding experiments show that it is cooperatively folded with a DeltaG degrees of unfolding of 1.8-2.0 kcal/mol and a T(m) of 58 degrees C. This peptide represents the first demonstration of the independent folding of an isolated split beta-alpha-beta motif, and is one of only four naturally occurring sequences of fewer than 40 residues that has been shown to fold cooperatively in the absence of disulfides or ligand binding.

Amino Acid Motifs↗

PubMed2002

Coupling of ligand binding and dimerization of helix-loop-helix peptides: spectroscopic and sedimentation analyses of calbindin D9k EF-hands.

Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range lgK = 4.6-5.3.

Amino Acid Sequence↗