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A Basic Understanding of Intrinsically Disordered Proteins
The document discusses intrinsically disordered proteins (IDPs), highlighting their structural characteristics and significant roles in biological processes such as regulation and disease. It notes a historical underestimation of protein disorder due to experimental biases and advancements in genomic data since the 1990s that helped in discovering IDPs. The document also outlines the implications of IDPs in various diseases, including cancer and Alzheimer’s, and aims to provide insights into their atomistic properties.
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A Basic Understanding of Intrinsically Disordered Proteins
- 1. Structural Understanding of IntrinsicallyDisordered Proteins Joe Passman April 26, 2013 6/24/20131
- 2. While I HaveYour Attention… 6/24/20132
- 3.
- 4. Rise, Prevalence, and PossibleRoles of Disorder in Proteins Background 6/24/20134
- 5. Structure / FunctionParadigm Transcription mRNA mRNA Translation Peptide 3D Protein Folding Output Nucleus DNA Splicing Export RNA ??? 6/24/20135
- 6. Disorder Becomes Apparent Early discovery Bovine serum albumin binding sites (Karush,1950) Later… Rapid rise of genomic data (~1990) Predictors of natural disordered regions (PONDRs) Early proton NMR experiments (Daniels et al, 1978) 0 200 400 600 800 2013 2010 2007 2004 2001 1998 1995 1992 1989 1986 1983 1978 Numberof Publications Year 6/24/20136
- 7. Disorder, Disorder, (most)Everywhere! Hosodaet al, 2011 6/24/20137
- 8. Why Did WeMiss It? Unobserved Bias of experiment Access to genomic data limited before ~1990 Crystal structure relatively uninformative Ignored Crystal structure artifacts dismissed Disorder thought to be an artifact Dyson & Wright, 2005 6/24/20138
- 9. What is Disorderin Proteins? Definition: A protein that does not adopt a well-defined native structure when isolated in solution under near- physiological conditions (Eliezer, 2009) 2 types Denatured state ensembles (DSEs) Intrinsically disordered proteins (IDPs) Vast and malleable configurational ensembles (CEs) Charged What can impact disorder? 6/24/20139
- 10. Why are IDPsInteresting? Diverse Roles! Regulatory Homeostasis of signaling pathways Translation/Transcription Structural Flexible Linkers AND….. They can kill you. Disease states Cancer (lack of cell cycle regulation) Brain (amyloid plaque formation) Lee et al, 2003 6/24/201310
- 11. Proposed Mechanisms 6/24/201311 Regulation Folding upon binding Highly specific / low affinity binding Multiple interaction sites Aggregation Dyson & Wright, 2005Schärpf et al, 2001
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- 13. Structure 6/24/201313 107 residues(1799 atoms) Positively charged side chains Proportion of arginine to lysine: 22% Structure Position Length (residues) Visual Helix 4-10 7 Helix 12-20 9 Helix 23-25 3 Beta Strand 26-29 4 Beta Sheet Alpha Helix
- 14. Function 6/24/201314 Transient complex Interacts with RNA RNA polymerase End Result Prevent termination of transcription Goldenberg, 2012
- 15. More on Interactionwith RNA 6/24/201315 Schärpf et al, 2001
- 16. 6/24/201316 Regulatory function Multiple interaction partners Extensively unfolded in isolation Flexible structure
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- 18. What Are WeTrying to Address? 6/24/201318 Goldenberg, 2011
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- 20. Goals 6/24/201320 Provide aset of atomistic properties Quantify correspondence with macroscopic ensemble- averaged experimental data Develop reference point for crowding studies
- 21. Context: Alzheimer’s Disease NIH/ National Institute on Aging 6/24/201321
Editor's Notes
- #3 Disorder is prevalent in the eukaryotic proteome and functions include participation in critical cellular control mechanisms (binding for termination activation / suppression), usage as flexible linkers, channel transport, etc.The importance of the 𝜆 𝑁 protein is that it provides a route for the suppression of the Rho protein during RNA polymerization through high specific, low affinity interactions.Importance of macromolecules in (thermo)dynamic aspects of foldingDisorder is prevalent in the eukaryotic proteome and functions include participation in critical cellular control mechanisms (binding for termination activation / suppression), usage as flexible linkers, channel transport, etc.The importance of the 𝜆 𝑁 protein is that it provides a route for the suppression of the Rho protein during RNA polymerization through high specific, low affinity interactions.Importance of macromolecules in (thermo)dynamic aspects of folding
- #4 Drive Home Points of Talk:Disorder is prevalent in the eukaryotic proteome and functions include participation in critical cellular control mechanisms (binding for termination activation / suppression) , usage as flexible linkers, channel transport, etc.The importance of the 𝜆 𝑁 protein is that it provides a route for the suppression of the Rho protein during RNA polymerization through high specific, low affinity interactions.Importance of macromolecules in (thermo)dynamic aspects of foldingDrive Home Points of Talk:Disorder is prevalent in the eukaryotic proteome and functions include participation in critical cellular control mechanisms (binding for termination activation / suppression) , usage as flexible linkers, channel transport, etc.The importance of the 𝜆 𝑁 protein is that it provides a route for the suppression of the Rho protein during RNA polymerization through high specific, low affinity interactions.Importance of macromolecules in (thermo)dynamic aspects of folding
- #6 Standard modelTranscription of DNA -> Splicing -> Movement through nuclear membrane -> Translation by tRNA-> Folding-> FunctionIs this model always true?Massive 3-D structure function relationship studies have been conducted examining a quantitative relationship (Hvidsten et al, 2009).BUT: They concluded a positive correlation because disorder and some functions, such as dimerization, cation channel activity, and transcriptoncoactivatorStandard modelTranscription of DNA -> Splicing -> Movement through nuclear membrane -> Translation by tRNA-> Folding-> FunctionIs this model always true?Massive 3-D structure function relationship studies have been conducted examining a quantitative relationship (Hvidsten et al, 2009).BUT: They concluded a positive correlation because disorder and some functions, such as dimerization, cation channel activity, and transcriptoncoactivator
- #7 Early discoveryKarush found a Gaussian-type description of binding site free energies for the serum albumin Ignored:Perhaps one reason was a belief that disorder is an artifact because protease digestion would eliminate such proteins in vivo. Later: Other indicators of disorder in the proteomeA series of PONDRs have been developed that take amino acid sequence inputs and give disorder tendency outputs (6, 9, 10, 13, 14). The various PONDRs are distinguished by training sets, data representations for their inputs, and machine learning models for their development.PONDRs may underestimate disorder in vivo due to the presence of the regions involved in ligand binding or crystal contacts.11% of a dataset containing more than 17 000 disordered residues from over 140 proteins were falsely predicted to be in correspondingly long ordered regions.Daniels et al performed and NMR study that found that the major part of the membrane network is the interaction of the chromogranin protein, shown to have approximately a random coil conformation, with the adenosine triphosphate and the adrenaline. Suggests that the purpose of the organized loose structure would appear to be to lower osmotic pressure without hindering rapid release of the vesicle contents on breaking the membraneNMR spectroscopy effective in identifying local structure
- #8 If disorder was a snake, it would have bitten usEukaryotes hypothesized to exhibit more disorder due to its strong correlation with regulatory and signaling functions.Some exceptions to complexity of organism -> more disorder Single-celled eukaryotes with host-changing lifestyleTypically > 30% of eucaryotic proteins have disordered regions of length > or = 50 consecutive residues (Dunker, 2001)Picture:Hsoda et al developed a Dichotomic System to Divide Proteins into Structural/un-structural Regions (DICHOT)system to classify the entire protein sequence into structural domains (SDs) and intrinsically disordered (ID) regions (Fukuchi, 2009).The new dichotomic system first identifies domains of known structures, followed by assignment of structural domains and ID regions with a combination of pre-existing tools and a newly developed program based on sequence divergence, taking un-aligned regions into consideration.Application of this system to human TFs (401 proteins) showed that 38% of the residues were in SDs, while 62% were in ID regions. The abundance of ID regions makes a sharp contrast to TFs of Escherichia coli (229 proteins), in which only 5% fell in ID regions.If disorder was a snake, it would have bitten usEukaryotes hypothesized to exhibit more disorder due to its strong correlation with regulatory and signaling functions.Some exceptions to complexity of organism -> more disorder Single-celled eukaryotes with host-changing lifestyleTypically > 30% of eucaryotic proteins have disordered regions of length > or = 50 consecutive residues (Dunker, 2001)Picture:Hsoda et al developed a Dichotomic System to Divide Proteins into Structural/un-structural Regions (DICHOT)system to classify the entire protein sequence into structural domains (SDs) and intrinsically disordered (ID) regions (Fukuchi, 2009).The new dichotomic system first identifies domains of known structures, followed by assignment of structural domains and ID regions with a combination of pre-existing tools and a newly developed program based on sequence divergence, taking un-aligned regions into consideration.Application of this system to human TFs (401 proteins) showed that 38% of the residues were in SDs, while 62% were in ID regions. The abundance of ID regions makes a sharp contrast to TFs of Escherichia coli (229 proteins), in which only 5% fell in ID regions.
- #9 No observationBias of experimentProcess of making homogenate of plant / animal tissue releases proteasesUnder these conditions, unfolded proteins more sensitive to proteolytic activityGenomic mappingSequence to structure mapping is difficult unless the sequence has a high level of homology to sequences with known structure.Even then… Not all mRNAs are actively translated at any particular time and IDPs are transient.Crystal structure uninformativeOnly indicates presence of disorder through absence of electron densityMay have bound ligand -> leads to induced orderMissing loops in structure indicateGaps may be alerting us to the presence of intrinsically disordered loopsSuch gaps are the basis for the DISOPRED2 disorder prediction serverIgnored Crystal structure artifactsThese "gaps" in the model are often thought to be artifacts of inadvertent disorder in the crystal.Artifactbecause protease digestion would eliminate such proteins in vivo.No observationBias of experimentProcess of making homogenate of plant / animal tissue releases proteasesUnder these conditions, unfolded proteins more sensitive to proteolytic activityGenomic mappingSequence to structure mapping is difficult unless the sequence has a high level of homology to sequences with known structure.Even then… Not all mRNAs are actively translated at any particular time and IDPs are transient.Crystal structure uninformativeOnly indicates presence of disorder through absence of electron densityMay have bound ligand -> leads to induced orderMissing loops in structure indicateGaps may be alerting us to the presence of intrinsically disordered loopsSuch gaps are the basis for the DISOPRED2 disorder prediction serverIgnored Crystal structure artifactsThese "gaps" in the model are often thought to be artifacts of inadvertent disorder in the crystal.Artifactbecause protease digestion would eliminate such proteins in vivo.
- #10 DSEs – normally foldIDPs – normally do not foldWhat can impact disorder?Changes in solution conditions
- #12 RegulationFolding upon bidning supported by chemical shifts in NMR spectra, especially for
- #14 Numerous specific and nonspecific contacts between the positively charged side-chains of these amino acids and the negatively charged phosphodiester backbone of the RNA are possible, giving a likely explanation for complex aggregation at relatively low concentrations of 300 µm.
- #15 Termination mechanism unknown
- #16 N-terminal segment binds to RNAC-terminal segment interacts with RNAP
- #19 The figure above is a cartoon representation of one of the unfolded proteins we study, the N protein of bacteriophage lambda (blue), in a solution containing about about 130 mg/mL of a small globular protein (orange). The blue sphere has the same volume as the unfolded protein. We are using small-angle neutron scattering (SANS) to study the behavior of unfolded proteins under conditions like those depicted in the figure, and we use computer simulations to test various ideas about unfolded proteins. One of the important results to emerge from these studies is the finding that crowding causes surprisingly little effect on the average dimensions of the N protein, and likely other unfolded proteins.
- #20 R_g from SAXS data