RVP-Lecture-2009 - Overview of protein structure, folding,...

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Unformatted text preview: Overview of protein structure, folding, and aggregation For an inventory of protein structures, visit the protein data bank at http:// www.rcsb.org 1 1 Outline 1. Introduction to proteins and their biological relevance 2. Overview of protein structure 3. Protein folding Driving forces 4. The biomedical relevance of protein folding Protein misfolding and aggregation in disease Avenues to pursue as a Biomedical Engineer in studying and alleviating the consequences of protein aggregation 2 2 Biological relevance of proteins Proteins are molecular machines, the building blocks, and arms of a living cell 3 3 Functions associated with proteins Major function as enzymes to catalyze chemical reactions Regulatory proteins control gene expression Receptor proteins accept intercellular signals that are transmitted by hormones, which are also proteins Immuno proteins recognize and bind “foe” molecules and “friendly” molecules Structural proteins form microfilaments, beams, girders, microtubules, as well as fibrils, hair, silk and protective coatings Transfer proteins transfer (molecules stored by storage proteins) Proteins are responsible for shuttling electrons and protons across the membrane 4 4 5 5 6 6 Introduction to protein structure 7 7 Relationship between structure and function The structure of a protein determines its function Generally the physico-chemical and geometric properties of protein surfaces determine specificity or protein activity Internal organization and protein architecture help fix the surface Function does not generally determine structure Proteins of similar overall structure can perform very different functions Function is only a loose determinant of structure – especially if the molecule that interacts with the protein is small 8 8 General structure of amino-acids SC H 2N CH CO2H 9 9 10 10 11 11 12 12 Peptide bond 13 13 Degrees of freedom 14 14 Driving forces 15 15 Interactions that stabilize the folded conformations of proteins 16 16 The importance of steric exclusion 17 17 Importance of hydrogen bonds 18 18 Hydrophobic hydration 19 19 Proteins “fold” spontaneously to their native three dimensional structures 20 20 Protein folding Refers to the process by which a linear sequence of amino acids adopts the well-defined three dimensional structure responsible for its biological function Within the cell, the native – biologically active – form of the protein appears to be adopted concomittantly with biosynthesis However, it does not appear that folding is unqiue to the goings on in vivo As it turns out Christian Anfinsen showed that the folded structure of a protein can be realized “spontaneously” upon renaturation – what does this mean? 21 21 The Anfinsen Experiment Take your favorite protein Stick it in a test tube with a mixture of denaturing reagants or elevate the temperature raise or lower the pH etc. i.e., generally insult the protein The protein responds by unfolding – this is assayed by testing for biological activity which will be absent or by spectroscopic probes which supports the notion that there is loss of the three-dimensional structure that is the hallmark of folded, native proteins Now restore the conditions to something that resembles the physiological milieu i.e., restore the pH, or remove the denaturant, lower the temperature etc. You will find that the protein is capable of refolding to its biologically active form i.e., all of the information required to fold is encoded in the amino acid sequence and the interactions that result between the sequence and the surrounding milieu 22 22 Anfinsen’s Thermodynamic Hypothesis “…the three-dimensional structure of a native protein in its normal milieu (solvent, pH, ionic strength, presence of other components such as metal ions or prosthetic groups, temperature and other) is the one in which the Gibbs free energy of the whole system is lowest; that is the native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment”. C.B. Anfinsen, Science (1973) 181: 223. 23 23 The protein folding problem has two parts… 1. 2. Why do proteins fold? Primarily a question about driving forces Of all the multitude of options, how does it come to be that conformations resembling the native structure is the (free) energetically preferred one under folding conditions To answer this question, we need knowledge of the native structure, the collection of non-native states, and the types of interactions that lead to a preference for one type of ensemble over another How do proteins fold? This is a question of rates and routes What type of barriers will a protein encounter as it seeks out the native state? What types of kinetic intermediates – pit stops – will a protein encounter as it folds up? 24 24 Why do proteins fold? Primarily solvent-driven: Of the multitude of conceivable conformations, a protein prefers those that minimize the interface between the surrounding aqueous milieu. This is achieved by adopting globular shapes whereby “oily” groups are partitioned to the interior of the globule Further stabilization is derived from the fact that proteins form tightly-packed interiors and the forces that hold the core together resist perturbations that try to unfold the protein When polar “non-oily” groups have to be buried, hydrogen bonds and salt-bridges provide further stabilization 25 25 Protein folding kinetics How fast do proteins fold? Why do protein folding times span roughly 12 orders of magnitudes i.e., why do different proteins have vastly different folding times? Some proteins fold rapidly without the accumulation of intermediates, whereas some others populate either kinetic intermediates or are confronted by off-pathway intermediates These intermediates are of the partially unfolded variety and they can get proteins in trouble – because they foster concentration-dependent interprotein interactions that lead to protein aggregation How does the cellular machinery exercise control over protein folding routes? If control is so exquisite, why then do proteins manage to go through partial unfolding and make deleterious inter-molecular interactions? These are questions that are the domain of basic molecular engineering, where important and challenging questions need to be asked about polymer dynamics and interactions in multi-component systems – the purview of thermodynamics and kinetics 26 26 Folding@Home – Copyright Vijay S. Pande (Stanford University) Loan your computer (screen saver) for something useful and learn about protein folding27 27 The Engineering Challenge A long-standing goal has been the design of “responsive” proteins which fold with “designed” stability profiles For example, you might want to design a protein that would be resistant to enzymatic activity in the cell or have a long shelf life so you can make a “drug” out of the protein This requires a robust strategy for predicting – accurately – the stability profiles of proteins and for using this ability to achieve specific design criteria You should note that although we have made significant advances, we are long way from being there It appears that the grand challenge (at least as I see it) is an inadequate knowledge of the types of non-native states a protein can adopt in a physiological milieu and more importantly, what the relative stabilities of these non-native states are vis-à-vis the native state 28 28 Protein aggregation is associated with disease Alzheimer’s disease Intracellular tangles – aggregates of MAP-Tau Extracellular neuritic plaques – aggregates of APP-A Parkinson’s disease Intracellular Lewy bodies – aggregates of -synuclein Prion diseases (scrapie, Mad Cow, Creutzfeld-Jacob) Extracellular amyloid-like deposits – aggregates of mutant or misfolded PrP Huntington’s and other CAG repeat diseases Intranuclear amyloid-like deposits – aggregates of proteins containing polyglutamine expansions 29 29 Production of Amyloid Amyloid precursor protein (APP), a transmembrane protein, is processed by two proteases. -secretase snips off a portion of the extracellular amino-terminal domain. Then, secretase cleaves APP, which liberates the amyloid- protein (A ). A accumulates to form long filaments (fibrils) of densely packed -pleated protein sheets. It is thought that these plaques of insoluble protein, or an intermediate form of the A in the pathway, are central to the pathogenesis of Alzheimer's disease. Cleavage of APP also produces another intracellular protein fragment, the APP intracellular domain (AICD). 30 -sheets: A common structural theme in aggregates 31 31 Amino acid sequence of A 42; Color code reflects the expected sidechain charge neat pH 7.0; (red is negative, blue is positive, and black is neutral) 32 Nucleated polymerization Nucleation-dependent polymerization, reflecting the unfavorable self-association of X natively folded monomers (in this case, six total) to form a fibril nucleus and the favorable addition of a large indeterminate number of monomers to the nucleus (nascent fibril) during fibril elongation 33 A belongs to the class of “intrinsically disordered” proteins, existing in the monomer state as an equilibrium mixture of many conformers. On-pathway assembly requires the formation of a partially folded monomer that self-associates to form a nucleus for fibril elongation, a paranucleus (in this case, containing six monomers). Fibril nucleation is unfavorable kinetically, which explains the lag phase of fibrillogenesis experiments, a period during which no fibril formation is apparent. Paranuclei selfassociate readily to form protofibrils, which are relatively narrow, short, flexible structures. These protofibrils comprise a significant but finite number (X) of paranuclei. Maturation of protofibrils through a process that is kinetically favorable yields classical amyloid-type fibrils. Other assembly pathways produce annular porelike structures, globular dodecameric (and higher order) structures, and amylospheroids. Annuli and amylospheroids appear to be off-pathway assemblies 34 Paradox In vitro - in test tube - concentrations of A alloforms that support aggregation, i.e., the saturation concentration is in the micromolar range In vivo, CSF / ISF levels of A 40 and A 42 tend to be in the picomolar range - data from Holzman’s group at WUSTL Why then do plaques form? Perhaps there are alternative mechanisms in play 35 35 Uptake of fluorescently labeled Abeta by neuroblastoma cells An acidic pH sensitive dye that identifies late endosomes / lysosomes Merged picture showing that taken up Abeta is coincident with location of acidic vesicles 36 Concentration of A 42 in vesicles is ca. 1000 fold higher than administered concentration 37 Implications and ongoing work Pathways exist to take up and concentrate A into acidic vesicles. Mechanism of uptake? We see aggregation of A 42 but not of A 40 in the vesicles - why? Competition between aggregation / degradation? Differences in kinetics of low pH aggregation? It is known that A 40 can inhibit aggregation of A 42. Is this in play? What happens to aggregates that are accumulated in intracellular vesicles? Do they end up outside cells by killing cells? If so how? And what are the structural characteristics of these aggregates? Using combinations of in silico modeling, in vitro experiments, in cell measurements, and in vivo (mouse) studies to sort these issues out 38 38 ...
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This note was uploaded on 02/14/2012 for the course NUBITRY 3304 taught by Professor Various during the Spring '01 term at Albertus Magnus.

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