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Biomedical subjects

Roger S Goody

Publications and source records attributed to Roger S Goody.

At least 19 recordsLinked to original sources
PubMed2006

Sec2 is a highly efficient exchange factor for the Rab protein Sec4.

Sec2 is a reversibly membrane associated multi-domain protein with guanine nucleotide exchange activity towards the yeast Rab-protein Sec4. Both proteins are localized to secretory vesicles destined for exocytosis. We have used transient kinetic methods to show that Sec2 is a highly active exchange factor, in contrast to other proteins previously characterized as Rab exchange factors. With a K(d) value for the Sec2:Sec4.GDP interaction of ca 70 microM and a maximal rate of GDP displacement of ca 15 s(-1), it is 100-1000-fold more effective than other proteins showing exchange activity towards Rabs (MSS4, DSS4, Vps9) and ca tenfold faster than Cdc25 as a Ras specific exchanger, although still 100-fold slower than the fastest systems studied so far, EF-Tu/Ef-Ts and Ran/RCC1. A comparison with other proteins showing Rab exchange activity shows that maximal rates of GDP dissociation catalyzed by Sec2 are orders of magnitude faster. When comparing Sec2 with DSS4, which also acts on Sec4, the difference was particularly dramatic. Another difference is seen in the kinetics of association of GTP with the Sec4:Sec2 complex, a process which is extremely slow for DSS4/MSS4 complexes with cognate Rabs but in the range observed for other GTPase:exchanger complexes for Sec4:Sec2., It is suggested that systems such as Ef-Tu/Ef-Ts and Ran/RCC1 have evolved for maximal possible activity for the interaction between two soluble proteins, whereas other evolutionary constraints which are connected to the spatial and temporal coordination of events in vesicular transport and other regulatory networks have determined the detailed kinetic properties of the other systems.

GTP-Binding Proteins↗
PubMed2006

Lipidated ras and rab peptides and proteins--synthesis, structure, and function.

Chemical biology can be defined as the study of biological phenomena from a chemical approach. Based on the analysis of relevant biological phenomena and their structural foundation, unsolved problems are identified and tackled through a combination of chemistry and biology. Thus, new synthetic methods and strategies are developed and employed for the construction of compounds that are used to investigate biological procedures. Solid-phase synthesis has emerged as the preferred method for the synthesis of lipidated peptides, which can be chemoselectively ligated to proteins of the Ras superfamily. The generated peptides and proteins have solved biological questions in the field of the Ras-superfamily GTPases that are not amendable to chemical or biological techniques alone.

Animals↗
PubMed2006

A phosphoryl transfer intermediate in the GTPase reaction of Ras in complex with its GTPase-activating protein.

The hydrolysis of nucleoside triphosphates by enzymes is used as a regulation mechanism in key biological processes. Here, the GTP hydrolysis of the protein complex of Ras with its GTPase-activating protein is monitored at atomic resolution in a noncrystalline state by time-resolved FTIR spectroscopy. At 900 ms, after the attack of water at the gamma-phosphate, there appears a H2PO4- intermediate that is shown to be hydrogen-bonded in an eclipsed conformation to the beta-phosphate of GDP. The H2PO4- intermediate is in a position where it can either reform GTP or be released from the protein in 7 s in the rate-limiting step of the GTPase reaction. We propose that such an intermediate also occurs in other GTPases and ATPases.

Binding Sites↗
PubMed2006

A generic building block for C- and N-terminal protein-labeling and protein-immobilization.

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.

Azides↗
PubMed2006

A newly designed microspectrofluorometer for kinetic studies on protein crystals in combination with x-ray diffraction.

We present a new design for a fluorescence microspectrophotometer for use in kinetic crystallography in combination with x-ray diffraction experiments. The FLUMIX device (Fluorescence spectroscopy to monitor intermediates in x-ray crystallography) is built for 0 degrees fluorescence detection, which has several advantages in comparison to a conventional fluorometer with 90 degrees design. Due to the reduced spatial requirements and the need for only one objective, the system is highly versatile, easy to handle, and can be used for many different applications. In combination with a conventional stereomicroscope, fluorescence measurements or reaction initiation can be performed directly in a hanging drop crystallization setup. The FLUMIX device can be combined with most x-ray sources, normally without the need of a specialized mechanical support. As a biological model system, we have used H-Ras p21 with an artificially introduced photo-labile GTP precursor (caged GTP) and a covalently attached fluorophore (IANBD amide). Using the FLUMIX system, detailed information about the state of photolyzed crystals of the modified H-Ras p21 (p21(mod)) could be obtained. Measurements in combination with a synchrotron beamline showed significant fluorescence changes in p21(mod) crystals even within a few seconds of x-ray exposure at 100 K.

Chromatography, High Pressure Liquid↗
PubMed2006

Nucleotide exchange via local protein unfolding--structure of Rab8 in complex with MSS4.

Rab GTPases function as essential regulators of vesicle transport in eukaryotic cells. MSS4 was shown to stimulate nucleotide exchange on Rab proteins associated with the exocytic pathway and to have nucleotide-free-Rab chaperone activity. A detailed kinetic analysis of MSS4 interaction with Rab8 showed that MSS4 is a relatively slow exchange factor that forms a long-lived nucleotide-free complex with RabGTPase. In contrast to other characterized exchange factor-GTPase complexes, MSS4:Rab8 complex binds GTP faster than GDP, but still ca. 3 orders of magnitude more slowly than comparable complexes. The crystal structure of the nucleotide-free MSS4:Rab8 complex revealed that MSS4 binds to the Switch I and interswitch regions of Rab8, forming an intermolecular beta-sheet. Complex formation results in dramatic structural changes of the Rab8 molecule, leading to unfolding of the nucleotide-binding site and surrounding structural elements, facilitating nucleotide release and slowing its rebinding. Coupling of nucleotide exchange activity to a cycle of GTPase unfolding and refolding represents a novel nucleotide exchange mechanism.

Amino Acid Sequence↗
PubMed2006

Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids.

Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.

4-Chloro-7-nitrobenzofurazan↗
PubMed2006

Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling.

In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Guanine nucleotide exchange factors operate by a simple allosteric competitive mechanism.

Guanine nucleotide exchange factors are essential components of the mode of action of GTPases involved in signal transduction. Their fundamental mechanism is generally accepted to derive from stabilization of the nucleotide-free form of GTPases, which is reflected in an increase in the rate of GDP dissociation when such an exchange factor is bound to a GTPase. The known kinetic properties of exchange factors can be explained on the basis of this simple allosteric competitive mechanism. Here, we describe experiments designed to distinguish this mechanism from a newer model, which invokes an active role for the incoming (i.e., displacing) nucleotide, implying the transient formation of a quaternary complex consisting of an exchange factor, a GTPase, and two nucleotides, one which is being displaced while the other stimulates this displacement. We show that for a well-known system (the small GTPase Ras and its exchange factor Cdc25) there is no evidence for an effect of the concentration or the nature (i.e., GDP or GTP) of the displacing nucleotide on the rate constant of GDP release from the Cdc25.Ras.GDP complex, consistent with the simple allosteric competitive model, and in disagreement with the newer suggestion. In addition, we present arguments, which demonstrate how the erroneous conclusions leading to the alternative model were derived.

Allosteric Regulation↗
PubMed2005

Hydrostatic pressure affects the conformational equilibrium of Salmonella typhimurium tryptophan synthase.

The effect of hydrostatic pressure on the tryptophan (Trp) synthase alpha2beta2 complex from Salmonella typhimurium has been investigated. Trp synthase has been shown previously to exhibit low-activity (open) and high-activity (closed) conformations. The equilibrium between the open and closed conformations of Trp synthase has been found to be affected by a wide range of variables, including alpha-subunit ligands, monovalent cations, organic solvents, pH, and temperature. The absorption spectrum of the Trp synthase-L-Ser complex shows an increase in absorption of the 423 nm band of the external aldimine, which is a characteristic of the open conformation, as hydrostatic pressure is increased from 1 to 2000 bar. The deltaV(o) and K(o) for the equilibrium between the closed and open conformations of the Trp synthase-L-Ser complex are -126 mL/mol and 0.12 for the Na+ form and -171 mL/mol and 2.3 x 10(-4) for the NH4+ form. When the Trp synthase-L-Ser complex is subjected to pressure jumps of 100-400 bar, relaxations are observed, exhibiting an increase in fluorescence emission at wavelengths greater than 455 nm, with 405 nm excitation. The relaxation to the new equilibrium position requires two exponentials to fit the data in the presence of 0.1 M Na+ and three exponentials to obtain a reasonable fit in the absence of cations and with 0.1 M NH4+. Fluorescence emission at 325 nm, with excitation at 280 nm, also increases when the Trp synthase-L-Ser complex is subjected to pressure jump. These data demonstrate that the open conformation of Trp synthase is favored by higher pressure. Thus, the open conformation has a smaller apparent net system volume than the closed conformation. We estimate that there are 35-47 more waters in the solvation shell of the open conformation than in that of the closed conformation.

Bacterial Proteins↗
PubMed2005

Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation.

Prenylated proteins with non-native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction, as the generated peptides could be ligated onto thioester-tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid-binding site.

Chromatography, Gel↗
PubMed2005

Chemical biology of protein lipidation: semi-synthesis and structure elucidation of prenylated RabGTPases.

Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells. For their function Rab/Ypt proteins require double modification with two covalently bound geranylgeranyl lipid moieties at the C-terminus. Generally, prenylated proteins are very difficult to obtain by recombinant or enzymatic methods. We generated prenylated RabGTPases using a combination of chemical synthesis and protein engineering. This semi-synthesis depends largely on the availability of functionalized prenylated peptides corresponding to the proteins' native structure or modifications. We developed solution phase and solid phase strategies for the generation of peptides corresponding to the prenylated C-terminus of Rab7 GTPase in preparative amounts enabling us to crystallize the mono-prenylated Ypt1:RabGDI complex. The structure of the complex provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins and a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.

Models, Molecular↗
PubMed2005

A genetically encodable microtag for chemo-enzymatic derivatization and purification of recombinant proteins.

Efficient separation of recombinant polypeptides from proteins of the expression host and their subsequent derivatisation with functional chemical groups is essential for the success of many biological applications. Numerous tag systems have been developed to facilitate the purification procedure but only limited progress has been made in development of generic methods for targeted modification of proteins with functional groups. In this work, we present a novel 6 amino acid long C-terminal protein tag that can be selectively modified with functionalized derivatives of farnesyl isoprenoids by protein farnesyltransferase. The reaction could be performed in complex protein mixtures without detectable unspecific labeling. We demonstrate that this modification can be used to purify the target protein by over 800-fold in a single purification step using phase partitioning. Moreover, we show that the fluorescent group could be used to monitor the interaction of the derivatized proteins with other polypeptides.

Electrophoresis, Polyacrylamide Gel↗