Robert Fox - US grants

The mechanism by which protein molecules are localized in various compartments and membranes of the cell has stood as one of the great problems of cell biology. It is now clear that nascent polypeptide chains are carried into the lumen of that organelle where the leader (signal) sequence is removed. Completed polypeptides travel to the Golgi body where they are targeted to specific organelles. Plasma membrane receptors such as the low density lipoprotein (LDL) receptor are cycled from the plasma membrane through coated endocytic vesicles and endosomes, delivering LDL particles to the lysosomes while the receptors return to the cell surface. In humans, errors in the targeting, processing and recycling of LDL receptors lead to familial hypercholesterolemia resulting in premature atherosclerosis. The general goals of this research program are to probe the physical and chemical basis by which membrane proteins attain and maintain defined three-demensional structures in biological membranes, the mechanism by which such proteins carry out their biological function and the role of electric fields in these processes. The best starting point for such investigations is a detailed three-dimensional structure derived from diffraction studies at a resolution consistent with atomic modeling. The specific aims of this research program are directed at two aspects of these general problems. First, the closely related membrane active peptides: alamethicin and suzukacillin will be studied as examples of proteins which can spontaneously integrate into lipid bilayers from aqueous solution. We plan to characterize the structure of these peptides in crystals grown from aqueous solution to define the molecular surface which interacts with the lipid bilayer. EM image reconstruction techniques will be applied to two dimensional arrays of gated and ungated peptide channels to define the structural basis of the voltage-gating process in these systems. Secondly, we plan to prepare three-dmensional crystals of E. coli leader peptidase, and to characterize its molecular structure at high resolution in an effort to define the structural basis of leader peptide recognition and cleavage, and the role of this process in membrane protein biogenesis.

The immediate goal of the proposed research is to learn more about the operating characteristics of the three principal phenomena of binocular vision--stereopsis, binocular rivalry, and binocular combination or fusion. Three related lines of inquiry are proposed, each dealing with one of the three principal phenomena. The method of investigation is the psychophysical paradigm, wherein considerable data are obtained from a relatively small number of human observers. The research on stereopsis is concerned with the way in which depth information is extracted from random-element stereograms and focuses upon the interaction of stereoscopic and physical contours, the contribution of cognitive variables to enhance the perception of stereopsis, the role of experience in perceiving stereopsis, and methods for assessing stereopsis in infants and young children. The research on binocular rivalry is designed to specify more precisely the kind of information that is lost during rivalry suppression and the relationship between rivalry suppression and the kind of suppression associated with binocular anomalies such as strabismus. The research on binocular combination attempts to test the hypothesis that suppression might be operative during the ostensibly cooperative fusion states of normal binocular viewing. The long-range objective of the program is to contribute information that would aid in the development of a general theory of binocular vision that would encompass stereopsis, rivalry, and binocular combination in a single framework. Moreover, the research has implications for both the etiology and the treatment of binocular visual anomalies in humans.

This research is concerned with the three major phenomena of binocular vision--stereopsis, rivalry, and fusion--and outlines investigations of these phenomena in adult humans, and in infants and young children using psychophysical or behavioral methods. The inquiry into stereopsis focuses on (a) sources of information about distance requisite for veridical perception of depth intervals, (b) differences in depth constancy between crossed and uncrossed disparities, (c) development of depth constancy, (d) the stereoscopic transfer function and its development, and (e) the interaction of motion and depth. Research on binocular rivalry examines the dichoptic interaction between higher order motion patterns (e.g., biological motion and vector motion) that permit an uncoupling of phenomenal conflict from the physical conflict between corresponding points normally present in rivalry-inducing stimuli. Research on binocular fusion investigates inhibitory interocular interactions, different from rivalry, that may be present during fusion. While the immediate objective is to learn more about each of these phenomena, a long range objective is to contribute to the development of a general theory of binocular vision that accommodates all within a unified framework.

Immunolglobulin molecules have become valuable reagents in research and diagnostic laboratories and have been tested as clinical reagents in the treatment of disease. Monoclonal antibodies directed to a linear epitope in a globular protein molecule can sometimes be generated by immunizing with linear peptide sequences derived from the native protein. When these antibodies are identified they are generally of low titer and thus not of great utility in the research laboratory or clinic. This is particularly true when this approach has been applied to molecules of the immunoglobulin superfamily. The limited success in generating antipeptide antibodies with a high affinity for the folded protein from which the sequence was derived may arise because the linear peptide conformation does not resemble the structure found in the folded protein. Two methods are proposed to constrain the conformation of linear epitopes in model immunogens. Sequences which occur on the surface of a parent protein in a Beta-turn conformation will be introduced into a cyclic peptide structure which should promote a Beta-turn conformation. These sequences will also be substitited for a Beta-turn region of staphylococcal nuclease at the gene level to produce a hybrid protein immunogen. The affinity of polyclonal and monoclonal antibodies, elicited by the model immunogens, for the parent protein will be compared with antibodies raised against similar linear peptides. The conformation of the cyclic peptides and hybrid proteins will be characterized by NMR spectroscopy and x-ray crystallagrophy to allow a detailed molecular interpretation of the immunological data.

Globular protein molecules are largely composed of beta-sheet and alpha- helical secondary structures separated by sharp changes in chain trajectory at four residue beta-turns or larger loops on the surface of the molecule. These betaturn and loop structures can often be recognized in protein sequences, aiding in secondary structure prediction and in the identification of potential linear peptide epitopes for antibody production. While antipeptide antibodies which recognize a full protein chain have been prepared in a number of cases, the resulting monoclonal antibodies often have a low affinity for the folded protein from which the synthetic peptide immunogen sequence was derived. A protocol was proposed to use a second protein (staphylococcal nuclease) as a "host" to constrain the "guest" peptide immunogen into a native structure by incorporating that sequence into a hybrid protein at an appropriate site. Work will continue on two hybrid protein systems to develop this method and to understand its success or failure in structural and physical terms. Results during the last three years indicate that there is a strong relationship between the sequence and type of a beta-turn which can be dominant over globular protein context effects. Investigations will continue to define the relationship between amino acid sequence and beta-turn type. These experiments should provide an improvement in our ability to predict betaturn sites in globular proteins and should define protein engineering design principles for the hybrid proteins above and other new protein molecules in general. NMR and x-ray crystallography experiments will continue in an effort to define the physical basis by which a cis peptide bond is favored in a type VI beta-turn of staphylococcal nuclease. This system provides an opportunity to observe the influence of amino acid sequence on the equilibrium between two beta-turn types on the surface of a globular protein. A genetic and crystallographic analysis will continue to define sequences consistent with a type I' beta-turn in nuclease, and the physical and structural basis for these sequences preferences.

DESCRIPTION: (Adapted From The Applicant's Abstract) This program of research investigates the ability of mildly mentally retarded adults to perceive and process correctly certain classes of stimuli with distinctive attributes. Included are global stereoscopic forms, global kinetic forms, patterns of biological motion and size/distance interactions produced by 3-D space. These stimuli engage preattentive processes early in the visual system and are seen effortlessly by nonretarded persons. However, prior research has demonstrated that such stimuli reveal substantial perceptual deficits in mildly retarded persons under conditions that eliminate, as contributing factors, peripheral visual anomalies and failures of comprehension. This is a surprising outcome because mild retardation is generally regarded to be a product of intellectual and cognitive deficiencies. The proposed inquiry seeks to specify more precisely the characteristics of these perceptual deficits. The resulting data should contribute directly to a deeper understanding of mental retardation. More generally they bear on the issue of the separability of cognitive and perceptual processes.

While many small proteins can refold spontaneously in vitro, the identification of the folding pathway, and the detection and structural characterization of folding intermediates have been difficult. Recently, attention has turned to the molten globule state and other nonnative equilibrium states of proteins which are thought to be models for kinetic intermediates in protein folding. The molten globule state of apomyoglobin can be populated at equilibrium at reduced Ph as demonstrated by a number of optical and hydrodynamic methods, and has recently been shown to represent a kinetic intermediate on the myoglobin folding pathway. Amide protection studies have identified that only the A, G, and H helices of myoglobin are protected in the molten globule state, suggesting that a native-like substructure involving those regions may persist. Fragments of staphylococcal nuclease have been produced which also have properties similar to those of the molten globule, i.e. a somewhat compact structure with some secondary structure but without a defined tertiary structure. Recently we have developed a chemical cleavage method where an EDTA-Fe based reagent (EPD) can be attached to a protein via a cysteine side chain. The addition of ascorbate generates hydroxyl radicals at the iron center which diffuse and cleave the polypeptide backbone in a region close to the cysteine attachment site at residues accessible to solvent. The observed cleavage sites can be mapped by amino acid sequencing. The cleavage is dependent on protein conformation. We propose to characterize the molten globule state of apomyoglobin and staphylococcal nuclease fragment structures using this newly developed chemical cleave technique. We will prepare a number of cysteine variants of these proteins and characterize the cleavage patterns observed in the native and molten globule states.

This program of research is concerned with the ability of persons with Prader-Willi syndrome (PWS) to perceive correctly certain classes of stimuli that have been found to pose substantial perceptual problems for adults with mild mental retardation. They include global stereoscopic forms generated by random element stereograms, global motion of kinetic forms generated by random element kinematograms, and the integration of stimulus dimensions that define the metric of three-dimensional space. While these stimuli are simple physically, they induce complex percepts. Moreover, they are perceived quickly and automatically without engaging attention or other cognitive processes. These characteristics have inspired efforts to develop models and computer simulations of the underlying processes. That inquiry has revealed that these stimuli impose formidable information processing or computational demands on the perceptual system. The computational problems they pose, led my colleagues and me to the conjecture that mildly retarded persons may encounter in perceiving them correctly. Considerable evidence in support of that conjecture has been obtained from a series of studies devoted to elucidating the perceptual capacities of mildly retarded adults. The basic result is that the mildly retarded persons can process the stimuli well above chance when they are at maximum values with respect to energy and information. But modest reduction in those variables produces substantial impairments in performance. Under conditions where nonretarded subjects perform without error, retarded subjects perform at chance. The pattern of results from this line of inquiry supports strongly the hypothesis that the heretofore unsuspected perceptual deficits reflect fundamental impairment of visual cortical mechanisms. The present project assesses the perceptual capacity of persons with PWS using the same stimulus conditions as those employed in the investigation of the mildly retarded. These include discrimination of forms defined by motion, with variations in density and correlation, and sensitivity to environment cues to visual space. The same psychophysical methods will be employed using equipment already in place.

DESCRIPTION: The overall goal of this proposal is the structure determination of recombinant human a1 glycine receptor chloride channel. The glycine receptor is part of a large family of ligand-gated channels that mediate signal transduction at the synapse. The structure of one member of this family, the acetylcholine receptor, has been studied in three dimensions by electron microscopy and a low resolution structure is available, however the sequence has not been mapped into this structure. Experiments are aimed at distinguishing plausible topological models for this family of channels by labeling cysteine residues engineered into the glycine receptor with chemical cleavage reagents. A major goal of the proposal is to crystallize purified holoreceptor as well as its extracellular, ligand binding domain. Functional glycine receptor composed of a single subunit type can be reconstituted from recombinant protein, and is produced in the co-P.I's lab in large enough yields for crystallization trials.

While may small proteins can refold spontaneously in vitro, the identification of the folding pathway, and the detection and structural characterization of folding intermediates have been difficult. Recently, attention has turned to the molten globule state and other nonnative equilibrium states of proteins which are thought to be models for kinetic intermediates in protein folding. Fragments of staphylococcal nuclease have been produced which also have properties similar to those of the molten globule, i.e. a somewhat compact structure with some secondary structure but without a defined tertiary structure. Pulsed hydrogen-deuterium exchange during refolding has been used to probe the protection of backbone amide hydrogens from solvent exchange during refolding of a number of proteins and most recently the staphylococcal nuclease Pro 117 yields Gly variant. The extent of exchange for 39 residues is determined by two-dimensional proton NMR after refolding for 5 ms to 10s. Three kinetic phases are inferred. Modest protection of amides in the early refolding intermediate composed to two beta-sheets formed by local sequence interactions is observed after a 5 ms refolding period. Native levels of protection throughout the molecule accrue more slowly in tow kinetic phases (k approximately 2s-1, k less than 0.01s-1). Protection factors were determined by varying the high pH labeling pulse after refolding for 100 ms. Little or no native or unfolded protein is present; instead, most molecules are in one or more partially folded states. The intermediate state has modest, yet significant, protection for residues in the beta-sheets (protection factors 10-60), and almost no protection in the alpha- helices (protection factors less than 10). The pattern of labeling is consistent with a role for beta turns and beta-hairpins in the formation of the early intermediate. Recently a chemical cleavage method has been developed where an EDT-Fe based reagent (EPD-Fe) can be attached to a protein via a cysteine side chain. The addition of ascorbate generates hydroxyl radicals at the iron center which diffuse and cleave the polypeptide backbone in a region close to the cysteine attachment site at residues accessible to solvent. The observed cleavage sites can be mapped by amino acid sequencing. The cleavage is dependent on protein conformation. We propose characterize the molten globule state of apomyoglobin and staphylococcal nuclease fragment structures using this newly developed chemical cleavage technique. We will prepare a number of cysteine variants of these proteins and characterize the cleavage patterns observed in the native and molten globule states.

[unreadable] DESCRIPTION (provided by applicant): Our previous studies have provided a proof-of-principle that a small disulfide-rich miniprotein can be developed that will block the infection of cells by Langat (LGT) virus, a naturally attenuated virus that is a model for the pathogenic members of the tick-borne encephalitis (TBE) serogroup of the Flavivirus genus. A first generation miniprotein, termed MP-100, was selected by panning a conformationally restrained combinatorial miniprotein phage display library for binding with purified recombinant domain III (D3) of the LGT virus envelope (E) protein. The miniprotein MP-100 was shown to block infection of Vero and LLC-MK2 monkey kidney cell cultures by tick-borne LGT and Powassan viruses. Further studies indicated an antiviral effect in a mouse animal model. Our objective during this period of support is the development of a second-generation, more tightly binding miniprotein with improved antiviral activity against TBE serogroup flaviviruses compared to the current MP-100 sequence. Our goal is to develop an antiviral miniprotein that is effective against a broad range of potential flavivirus bioterrorist threat agents in the TBE serogroup, including Central European tick-borne encephalitis (strain Kumlinge), Kyasanur Forest Disease (FD), Omsk Hemorrhagic Fever (OHF) and Russian Spring Summer encephalitis (RSSE) viruses. We will identify optimized tight-binding analogs of MP-100 to OHF-E-D3 and the related TBE serogroup E-D3s, and determine if they have enhanced antiviral activity in tissue culture cells and in animals. The development of anti-TBE virus miniproteins will serve as a model for the development of miniproteins against other flaviviruses that are also potential bioterrorist threat agents or emerging diseases, including dengue, Japanese encephalitis and West Nile. [unreadable] [unreadable]