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Biomedical subjects

C L Brooks

Publications and source records attributed to C L Brooks.

At least 19 recordsLinked to original sources
PubMed2006

ARF-BP1 as a potential therapeutic target.

In this review, we discuss the recent identification of ARF-BP1 (also known as Mule, UREB1, E3(histone), LASU1, and HectH9). ARF-BP1, a HECT domain-containing E3 ubiquitin ligase, interacts with ARF and p53. Its ubiquitin ligase activity is inhibited by ARF. Inactivation of ARF-BP1 stabilised p53 and induced apoptosis. Notably, inactivation of ARF-BP1 also caused cell growth repression in p53-null cells and breast cancer cells with mutant p53. Thus, ARF-BP1 emerges as a novel therapeutic target against cancer regardless of p53 status.

Cell Division↗
PubMed2006

Effects of an S84E mutation of bovine growth hormone in transgenic mice.

The ability of mutant bovine growth hormones (bGH) to serve as either agonist or antagonist has been demonstrated in transgenic mice. We have prepared two transgenic strains of FVB/N mice, one expressing wild-type bGH and a second with a glutamic acid mutation at serine 84 in helix 2. Comparison of their phenotypes to those of nontransgenic littermates indicates that wild-type bGH induces a previously described phenotype for hyper-somatotrophic mice. In contrast, the replacement of the side chain hydroxyl at serine 84 with acetic acid produced a phenotype that expressed bGH at appreciable concentrations, but failed to elicit the phenotype observed with either an agonist or an antagonist of bGH. These results indicate that serine 84 is crucial for the activity of bGH despite this site being distal to the receptor binding surfaces.

Acetic Acid↗
PubMed2004

Different elements of mini-helix 1 are required for human growth hormone or prolactin action via the prolactin receptor.

Human growth hormone (hGH) and prolactin (hPRL) have a low sequence homology, but both bind and activate hPRL receptors. hGH also binds hGH receptors. hGH has 22 and 20 kDa forms; residues 32-46 have been deleted by alternative RNA splicing to create the smaller form. hGH requires F44 for activity through the hPRL receptor, but not for activity through the hGH receptor. The deletion of F44 from hGH has the same effect as removal of residues 32-46 (approximately 200-fold loss in activity), indicating the importance of F44 in hGH when activating the hPRL receptor. In contrast, when the homologous F50 is deleted from hPRL little or no activity is lost, indicating that this highly conserved phenylalanine is not required for the action of hPRL. Deletion of residues 41-52 (a non-conserved sequence homologous to residues 32-46 of hGH) reduced the activity of hPRL by >14 000-fold. This region is essential for the biological activity of hPRL. As these two proteins have evolved from a common ancestor, they have retained the requirement for this region but need different structural elements to activate hPRL receptors. Such diversity represents an opportunity to fine-tune hormone activity.

Amino Acid Sequence↗
PubMed2003

Functional relationship of serine 90 phosphorylation and the surrounding putative salt bridge in bovine prolactin.

The biological activity of bovine prolactin (PRL) is reduced by in vivo phosphorylation of serine 90 (S90) that is located within a putative N+4 salt bridge (R89 and D93). We substituted hydrophobic, polar, or acidic residues for S90 and/or replaced members of the putative R89/D93 salt bridge to determine if a functional relationship between the putative salt bridge and the phosphorylation could be observed. At position 90 the bulk of the residue was the most important factor in modulating biological activity in either the rat Nb2 cell bioassay or PRL receptor binding. Charge played a smaller role. Replacement of either partner of the salt bridge reduced both biological and binding activities indicating the presence of a salt bridge at this position. The combination of replacing a salt bridge member and substituting glutamic acid at S90 produced greater than additive changes in our experimental endpoints, indicating a functional coupling between the salt bridge and phosphorylation site. We interpret the data to indicate that either in vivo phosphorylation or specific mutations that destabilize the salt bridge impairs biological activity.

Amino Acid Substitution↗
PubMed2003

Differential effects of zinc on functionally distinct human growth hormone mutations.

Human growth hormone (hGH) binds and activates lactogenic receptors by a sequential receptor dimerization mechanism. The affinity for the first lactogenic receptor is increased due to one zinc molecule linking hGH residues H18 and E174, located in helices 1 and 4, respectively, with two adjacent residues in the lactogenic receptor (D187 and H188). Two functionally unique groups of mutant hGHs have been identified. Addition of 25 microM zinc to lactogenic bioassays differentially affects mutant activities based on which group they belong to. One mutation (G120R) is located within the binding surface of hGH that interacts with the second lactogenic receptor. In the presence of endogenous zinc, G120R reduces the maximal activity of hGH without altering either the agonist or antagonist phases of the bell-shaped dose-response curve. Addition of zinc to this assay further reduces the activity of this protein. In contrast, mutations within a hydrophobic motif in hGH that functionally couples the two lactogenic receptor binding sites decrease the sensitivity (right-shift) of the agonist phase of the dose-response curve without similarly affecting the antagonist phase. The addition of zinc to these lactogenic assays increases the sensitivity (left-shifts) of the dose-response curves, largely negating the effect of these mutations. The effects of zinc differentiate between mutations within these two distinct functional motifs by limiting the pool of potential conformations that are available for binding within either of the receptor binding sites of this ligand.

Amino Acid Substitution↗
PubMed2001

Ligand-protein database: linking protein-ligand complex structures to binding data.

In computational structure-based drug design, the scoring functions are the cornerstones to the success of design/discovery. Many approaches have been explored to improve their reliability and accuracy, leading to three families of scoring functions: force-field-based, knowledge-based, and empirical. The last family is the most widely used in association with docking algorithms because of its speed, even though such empirical scoring functions produce far too many false positives to be fully reliable. In this work, we describe a World Wide Web accessible database that gathers the structural information from known complexes of the PDB with experimental binding data. This database, the Ligand-Protein DataBase (LPDB), is designed to allow the selection of complexes based on various properties of receptors and ligands for the design and parametrization of new scoring functions or to assess and improve existing ones. Moreover, for each complex, a continuum of ligand positions ranging from the crystallographic position to points on the surface of the protein receptor allows an assessment of the energetic behavior of particular scoring functions.

Databases, Factual↗
PubMed2001

Modeling of the metallo-beta-lactamase from B. fragilis: structural and dynamic effects of inhibitor binding.

The structure and dynamics of an inhibitor-bound complex of the metallo-beta-lactamase from Bacteroides fragilis are studied by using molecular dynamics. A search of the conformational space was performed to obtain three distinct models of the complex, which were then subjected to solvated molecular dynamics. A solvated molecular dynamics study of the apo protein was performed to serve as a baseline for comparison with the bound simulations. We find loop conformation changes due to binding as well as a decrease in flexibility of the protein as a whole and especially in the major loop of the beta-lactamase. We report the structural and dynamical features of the inhibitor-bound and apo models, as well as experimentally measurable quantities, which should be capable of distinguishing the two binding modes we have determined.

Apoproteins↗
PubMed2001

Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.

The residue Asp87, which is in the calcium-binding loop of bovine alpha-lactalbumin (alpha-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A alpha-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648-11654]. On the contrary, D87N alpha-LA displayed native-like secondary structure with a somewhat de-stabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N alpha-LA [Veprintsev et al. (1999) PROTEINS: Struct. Funct. Genet., 37, 65-72], the structure appeared to more closely resemble native alpha-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N alpha-LA was approximately 31 degrees C versus native apo- alpha-LA (approximately 25 degrees C), probably due to negative charge 'compensation' in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N alpha-LA was approximately 57 degrees C versus native alpha-LA (approximately 66 degrees C), which was related to a decreased Ca(II) affinity (K = approximately 2.1 x 10(5) versus approximately 1.7 x 10(7)/M at 40 degrees C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.

Animals↗
PubMed2001

From folding theories to folding proteins: a review and assessment of simulation studies of protein folding and unfolding.

Beginning with simplified lattice and continuum "minimalist" models and progressing to detailed atomic models, simulation studies have augmented and directed development of the modern landscape perspective of protein folding. In this review we discuss aspects of detailed atomic simulation methods applied to studies of protein folding free energy surfaces, using biased-sampling free energy methods and temperature-induced protein unfolding. We review studies from each on systems of particular experimental interest and assess the strengths and weaknesses of each approach in the context of "exact" results for both free energies and kinetics of a minimalist model for a beta-barrel protein. We illustrate in detail how each approach is implemented and discuss analysis methods that have been developed as components of these studies. We describe key insights into the relationship between protein topology and the folding mechanism emerging from folding free energy surface calculations. We further describe the determination of detailed "pathways" and models of folding transition states that have resulted from unfolding studies. Our assessment of the two methods suggests that both can provide, often complementary, details of folding mechanism and thermodynamics, but this success relies on (a) adequate sampling of diverse conformational regions for the biased-sampling free energy approach and (b) many trajectories at multiple temperatures for unfolding studies. Furthermore, we find that temperature-induced unfolding provides representatives of folding trajectories only when the topology and sequence (energy) provide a relatively funneled landscape and "off-pathway" intermediates do not exist.

Models, Molecular↗
PubMed2000

Enhanced immunogenicity of a conformational epitope of human T-lymphotropic virus type 1 using a novel chimeric peptide.

The ability of a peptide vaccine derived from the human T-lymphotropic virus type 1 (HTLV-1) surface envelope glycoprotein protein (gp46) to mimic the native protein and elicit a protective immune response has been examined. This peptide construct, designated MVFMF2, comprises amino acids (aa) 175-218 of gp46 linked by a four residue turn (GPSL) to a promiscuous T-cell epitope from the measles virus fusion protein (MVF, aa 288-302). The peptide was structurally characterized by circular dichroism (CD) spectroscopy and was found to contain alpha-helical secondary structure. The immunogenicity of MVFMF2 in rabbits and mice was evaluated by direct ELISA and competitive ELISA using peptide constructs and the recombinant protein ACH-RE3 (aa 165-306). This peptide, when administered with adjuvant (N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine, nor-MDP) was immunogenic in an outbred population of both rabbits and mice. Furthermore, the peptide construct was encapsulated in biodegradable microspheres of poly(D,L-lactide-co-glycolide) to eliminate booster immunization and to examine adjuvant requirements. The data indicate that MVFMF2 shows enhanced immunogenicity when encapsulated in biodegradable microspheres. Inoculation of the encapsulated peptide produced a similar humoral response to that of the free peptide, but did not require the use of adjuvant. Elicited anti-rabbit and anti-mouse antibodies recognized whole viral preparations and the recombinant protein ACH-RE3 in ELISA assays. Additionally, inoculated rabbits exhibited enhanced reactivity to viral antigens by western blot compared to non-vaccinated controls. Although anti-rabbit and anti-mouse antibodies were capable of inhibiting syncytium formation at low dilutions, rabbits were not protected from cell-associated viral challenge. Future development of vaccines to HTLV-1 may need to incorporate the ability to elicit cell-mediated immune responses in order to protect against cell-associated viral infection.

Amino Acid Sequence↗
PubMed2000

Accurate reconstruction of all-atom protein representations from side-chain-based low-resolution models.

A procedure for the reconstruction of all-atom protein structures from side-chain center-based low-resolution models is introduced and applied to a set of test proteins with high-resolution X-ray structures. The accuracy of the rebuilt all-atom models is measured by root mean square deviations to the corresponding X-ray structures and percentages of correct chi(1) and chi(2) side-chain dihedrals. The benefit of including C(alpha) positions in the low-resolution model is examined, and the effect of lattice-based models on the reconstruction accuracy is discussed. Programs and scripts implementing the reconstruction procedure are made available through the NIH research resource for Multiscale Modeling Tools in Structural Biology (http://mmtsb.scripps.edu).

Models, Molecular↗
PubMed2000

Zinc binding in bovine alpha-lactalbumin: sequence homology may not be a predictor of subtle functional features.

alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution.

Amino Acid Substitution↗
PubMed2000

The species specificity of growth hormone requires the cooperative interaction of two motifs.

Primate growth hormones (GH) activate both primate and non-primate somatotrophic receptors (GH receptors), but non-primate GHs do not activate primate GH receptors. Previous studies argued the interaction of Asp(171) of human GH and Arg(43) of the receptor produced an attractive ionic interaction. In non-primate GHs, His(170) replaces the homologous Asp(171), producing a repulsive interaction with Arg(43) of the primate receptor which was believed to reduce the attraction of non-primate GH for the human GH receptor, thus providing species specificity. In this report, H170D bovine GH had activity and affinity for human GH receptors approaching those of human GH. In contrast, replacing Asp(171) of human GH with His did not significantly reduce somatotrophic activity, indicating that species specificity is not wholly explained by this residue's interaction with Arg(43) of the receptor. Deletion of either Phe(44) (a residue present only in primate GHs) or residues 32-46 (20-kDa form of human GH) each only marginally reduced somatotrophic activities. But the combination of the D171H mutation with either DeltaPhe(44) or Delta32-46 in human GH reduced binding and activity in a greater than additive fashion, indicated a functional interaction between these distant structural features. In bovine GH addition of phenylalanine at position 44 increased the somatotrophic activity and receptor affinity in cells containing the human GH receptor. The combination of the H170D mutation and the addition of phenylalanine at position 44 created a bovine GH with activity indistinguishable from wild-type human GH. Based on evidence from both bovine and human GHs, the cooperative interaction of these two distant motifs determined the species specificity and indicated that structural plasticity was a critical feature necessary for the species specificity of somatotrophic activity.

Animals↗
PubMed1999

Exploring the origins of topological frustration: design of a minimally frustrated model of fragment B of protein A.

Topological frustration in an energetically unfrustrated off-lattice model of the helical protein fragment B of protein A from Staphylococcus aureus was investigated. This G-type model exhibited thermodynamic and kinetic signatures of a well-designed two-state folder with concurrent collapse and folding transitions and single exponential kinetics at the transition temperature. Topological frustration is determined in the absence of energetic frustration by the distribution of Fersht phi values. Topologically unfrustrated systems present a unimodal distribution sharply peaked at intermediate phi, whereas highly frustrated systems display a bimodal distribution peaked at low and high phi values. The distribution of phi values in protein A was determined both thermodynamically and kinetically. Both methods yielded a unimodal distribution centered at phi = 0.3 with tails extending to low and high phi values, indicating the presence of a small amount of topological frustration. The contacts with high phi values were located in the turn regions between helices I and II and II and III, intimating that these hairpins are in large part required in the transition state. Our results are in good agreement with all-atom simulations of protein A, as well as lattice simulations of a three- letter code 27-mer (which can be compared with a 60-residue helical protein). The relatively broad unimodal distribution of phi values obtained from the all-atom simulations and that from the minimalist model for the same native fold suggest that the structure of the transition state ensemble is determined mostly by the protein topology and not energetic frustration.

Kinetics↗
PubMed1999

Fine tuning the N-terminus of a calcium binding protein: alpha-lactalbumin.

The effects of amino acid substitutions in the N-terminus of bovine recombinant alpha-lactalbumin (including enzymatic removal of the N-terminal methionine and deletion of Glu-1) were studied by intrinsic fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC). Wild-type recombinant alpha-lactalbumin has a lower thermostability and calcium affinity compared to the native protein, while the properties of wild-type protein with the N-terminal methionine enzymatically removed are similar to the native protein. Taken together, the fluorescence, CD, and DSC results show that recombinant wild type alpha-lactalbumin in the absence of calcium ion is in a type of molten globule state. The delta-E1 mutant, where the Glu(1)residue of the native sequence is genetically removed, leaving an N-terminal methionine in its place, shows almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. It was concluded that the N-terminus of the protein dramatically affects both stability and function as manifested in calcium affinity. Proteins 1999;37:65-72.

Animals↗
PubMed1999

Methodology for protein-ligand binding studies: application to a model for drug resistance, the HIV/FIV protease system.

A protocol for the rapid energetic analysis of protein-ligand complexes has been developed. This protocol involves the generation of protein-ligand complex ensembles followed by an analysis of the binding free energy components. We apply this methodology toward understanding the origin of binding specificity within the human immunodeficiency virus/feline immunodeficiency virus (HIV/FIV) protease system, a model system for drug resistance studies. A distinct difference in the internal strain of an inhibitor within each protein environment clearly favors the HIV protease complex, as observed experimentally. Our analysis also predicts that residues within the S2-S3 pockets of the FIV protease active site are responsible for this strain. Close examination of the active site residue contributions to interaction energy and desolvation energy identifies specific amino acids that may also play a role in determining the binding preferences of these two enzymes. Proteins 1999;36:318-331.

Animals↗