Protein Structure, Post Translational Modifications and Protein Folding
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Post-translational modifications (PTMs) are covalent processing events that change the properties of a protein by proteolytic cleavage or by addition of a modifying group to one or more amino acids. Protein post-translational modification (PTM) plays an essential role in various cellular processes that modulates the physical and chemical properties, folding, conformation, stability and activity of proteins, thereby modifying the functions of proteins
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Protein Structure, Post Translational Modifications and Protein Folding
- 1. 2/8/2019 1Department of Plant Biotechnology Protein Structure, Post Translational Modifications and Protein Folding Suresh H. Antre 1st Year Ph. D. Plant Biotechnology
- 2. 2/8/2019 Department of Plant Biotechnology 2 Proteins are the major functional molecules in living cells, playing essential roles in various cellular processes such as catalysis, transport, and structural integrity. The human genome is estimated to harbor approximately 25,000 genes, alternative splicing of transcripts and post-translational modifications (PTMs) of proteins result in millions of proteins with diverse functions. PTMs regulate a protein’s function, level, and activity through the covalent attachment of small chemical molecules to certain amino acid residues, allowing proteins to respond to developmental signals or environmental stimuli.
- 3. 2/8/2019 Department of Plant Biotechnology 3 Protein modifications related to the regulation of biological processes
- 4. 2/8/2019 Department of Plant Biotechnology 4 Protein Structure
- 5. 2/8/2019 Department of Plant Biotechnology 5 Post-translational modifications (PTMs) are covalent processing events that change the properties of a protein by proteolytic cleavage or by addition of a modifying group to one or more amino acids. Protein post-translational modification (PTM) plays an essential role in various cellular processes that modulates the physical and chemical properties, folding, conformation, stability and activity of proteins, thereby modifying the functions of proteins. Post-translational modifications (PTMs)
- 6. 2/8/2019 Department of Plant Biotechnology 6 PTMs
- 7. 2/8/2019 Department of Plant Biotechnology 7 Some common and important post-translational modifications MARCH 2003 • VOLUME 21 • nature biotechnology
- 8. 2/8/2019 Department of Plant Biotechnology 8 Some common and important post-translational modifications MARCH 2003 • VOLUME 21 • nature biotechnology
- 9. 2/8/2019 9Department of Plant Biotechnology Posttranslational modification structural database (PTM-SD) http://www.dsimb.inserm.fr/dsimb_tools/PTM-SD/
- 10. 2/8/2019 Department of Plant Biotechnology 10 Database, Vol. 2014
- 11. 2/8/2019 Department of Plant Biotechnology 11 Structural and Functional annotations Experimental protein modifications BMC Research Notes 2009
- 12. 2/8/2019 Department of Plant Biotechnology 12 Protein folding is the process that leads from the linear amino acid sequence of a polypeptide chain to a defined spatial structure characteristic for the native protein. It is initiated by collapse of the polypeptide chain, which is driven by the “desire” of hydrophobic amino acids to escape the polar solvent water. Glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). Protein folding is however a very fast process within the range of seconds or even milliseconds. Protein Folding
- 13. 2/8/2019 Department of Plant Biotechnology 13 History The modern history of the protein folding problem began almost 50 years ago with the demonstration by Anfinsen and co-workers that ribonuclease A (RNase A) can fold with no help from other biological machinery (Anfinsen et al. 1961; Anfinsen, 1973). Anfinsen showed that, as for any chemical reaction, the folding of RNase A proceeds spontaneously downhill to the lowest free-energy polypeptide conformation, the predestined functional native state. Won the Nobel Prize for demonstrating that protein folding is governed solely by the protein itself. Other scientists discovered that some proteins have helped in the process - chaperones
- 14. 2/8/2019 Department of Plant Biotechnology 14 • Alzheimer’s disease • Cystic fibrosis • Mad Cow disease • Even many cancers Recent discoveries show that all these apparently unrelated diseases result from protein folding gone wrong.
- 15. 2/8/2019 Department of Plant Biotechnology 15 Initially it was proposed that proteins have to pass along specific folding pathways to find the native structure within a very short time (Energy Landscapes and Folding Funnels) Assumes that a protein's native state corresponds to its free energy minimum. Based on the physics concept of minimizing free energy. In the course of the folding process intermediates may accumulate that have hydrophobic amino acid side chains still exposed; these can serve as sticky surfaces and promote aggregation. Error-prone process, especially in the case of large proteins built up of several domains. (http://www.nature.com/nsb ) Protein folding mechanisms
- 16. 2/8/2019 Department of Plant Biotechnology 16 Key Findings _ The correct formation of disulfide bridges is especially important for secretory proteins containing cysteines. Folding intermediates with wrong conformations of peptidyl ― prolyl bonds or wrong disulfide bonds are also in danger of aggregating. Inside the cell, both of these folding reactions are therefore catalyzed by specific enzymes: PPIases and protein disulfide isomerases.
- 17. 2/8/2019 Department of Plant Biotechnology 17 An E. coli cell utilizes up to major protein that cross-links to virtually all nascent 20,000 ribosomes to produce an estimated total of 30,000 polypeptides per minute. The polypeptide exit channel was marked by tungsten cluster compounds bound in one heavy- atom-derivatized crystal. When does the process of folding begin in the lifetime of a protein? Crystallographic data of bacterial ribosomes identified a peptide exit tunnel in the large subunit with a length of 100 A and an average diameter of about 20 A (Ban et al., 1999).
- 18. 2/8/2019 Department of Plant Biotechnology 18 Folding inside the Cell Proteins are synthesized in vivo at the ribosomes in a vectorial manner from the N to the C terminus. They are either secreted or they fold into their native structure. During these stages proteins are in danger of undergoing misfolding and aggregtion. Even when proteins have already reached their native conformation they are vulnerable to misfolding due to Brownian motion especially under heat stress. Molecular Crowding
- 19. 2/8/2019 Department of Plant Biotechnology 20 De novo Protein Folding / The Welcoming Committee for Nascent Polypeptide Chains Newly synthesized proteins in bacteria associate with the chaperone trigger factor (TF), as soon as they leave the exit tunnel of the ribosome. So far the trigger factor has not been found in the cytosol of eukaryotic cells, but it is assumed that other proteins, like the ribosome-associated protein complex NAC, can take over trigger factor's function. Exposed hydrophobic side chains of the newly synthesized polypeptides are most likely protected from wrong interactions by association with the trigger factor. In addition, the trigger factor may prevent premature folding until a complete domain has emerged from the ribosomal exit site.
- 20. 2/8/2019 Department of Plant Biotechnology 21 Cell, Vol. 101, 119–122, April 14, 2000 Prokaryotes Eukaryotes Model for Folding of Newly Synthesized Proteins in Cytosol
- 21. 2/8/2019 Department of Plant Biotechnology 22 Molecular Chaperones Control Protein Folding To optimize cellular protein folding, protective systems have developed in the course of evolution. They assist the de novo folding of proteins or they form repair machines for misfolded or even aggregated proteins, and they are therefore especially important for the survival of cells during stress situations. Since heat shock can induce the synthesis of many chaperones, those are also called heat shock proteins(Hsps). The name of each of the chaperone families is derived from the molecular weight of the corresponding main representative (for example, Hsp70–a protein with a molecular weight of 70 kDa).
- 22. 2/8/2019 Department of Plant Biotechnology 23 Chaperone families: Structure and function
- 23. 2/8/2019 Department of Plant Biotechnology 24 Chaperone families: Structure and function
- 24. 2/8/2019 Department of Plant Biotechnology 25 How chaperones repair nascent or misfolded proteins . . . ? Stress conditions like a sudden increase in temperature can cause proteins to unfold and aggregate. Different chaperone systems function in the cell as a protective system to prevent protein aggregation by binding to the misfolded proteins. E.g. : DnaK (Hsp70) and GroEL (Hsp60) systems working together with regulatory co- chaperones like sHsps Nascent or misfolded polypeptides expose these segments and are therefore bound by chaperones. Thereby the association of several segments of different polypeptides, which would otherwise lead to aggregation, is prevented. This activity of chaperones is called holder function and can be exerted independently of adenosine triphosphate (ATP).
- 25. 2/8/2019 Department of Plant Biotechnology 26 Refolding into the active 3D structure requires the folder function, which is ATP- dependent. Two chaperone machines acting as folders have already been well studied: Hsp60 and Hsp70. Cntd . . .
- 26. 2/8/2019 Department of Plant Biotechnology 27 The Hsp60 chaperone The barrel shaped GroEL chaperone that is found in all prokaryotes, mitochondria, and chloroplasts consists of two stacked rings of seven subunits each. Each of the two GroEL rings forms a cavity in which misfolded polypeptides bind. Both rings fold substrates with a phase shift (green substrate, I-VI; blue substrate, IV-III) and each cycle takes about 15 seconds.
- 27. 2/8/2019 Department of Plant Biotechnology 28 Chaperones assist newly synthesized proteins to fold
- 28. 2/8/2019 Department of Plant Biotechnology 29 The Hsp70 chaperone In contrast to GroEL, DnaK does not enclose its substrate completely but only binds to a single short peptide segment of about five amino acids. Substrate binding and release is controlled by ATP hydrolysis to adenosine diphosphate (ADP) and ADP/ATP exchange processes, which are regulated by the co- chaperones DnaJ and GrpE. The cycles of substrate binding and release are, according to recent calculations, very short (about 1 second per cycle) and most likely have to be repeated many times until a protein is refolded.
- 29. 2/8/2019 Department of Plant Biotechnology 30 Chaperones assist newly synthesized proteins to fold
- 30. 2/8/2019 Department of Plant Biotechnology 31 However, it is not yet clear how these binding cycles promote the refolding of misfolded proteins. According to one hypothesis DnaK unfolds the substrate locally in contrast to the global unfolding by GroEL. This mechanism evidently has the big advantage that it is independent of the size of the misfolded protein substrate. It was shown that proteins of all sizes are found among the Hsp70 substrates and that proteins larger than 60 kDa are especially dependent on Hsp70 assistance during their folding.
- 31. 2/8/2019 Department of Plant Biotechnology 32 Summary Proteins are the major functional molecules in living cells, playing essential roles in various cellular processes. Common PTMs and their functions. Importance of protein folding pathways, factors and role of chaperons.
Hinweis der Redaktion
- Posttranslational modification structural database (PTM-SD) provides access to structurally solved modified residues, which are also experimentally annotated as PTMs. It gives valuable information on PTMs in the context of global and local protein conformation, and also particular details for each PTM observed in protein 3D structure. Four different databanks are used to generate the data. The protein structures are taken from PDB (17), while PTMs annotations are extracted from dbPTM (14) and PTMCuration (20). UniProt sequences (21) are aligned against the extracted PDB sequences. Thus, we obtained protein structures with PTM annotations and modified residues at exact same positions. At last a semantic mining was made to accept or not the correspondence between the modifications and the annotations.
- Figure 1. Folding funnels. A folding funnel represents the free energy of all potential protein structures as a function of the possible conformations. Different unfolded species of a protein ™roll down∫ the surface of the folding funnel. A folding funnel contains many local minima, in which a protein can fall during the folding process. Some local minima represent productive folding intermediates, with a stable and native like structure (molten globules), while others act like a trap and keep the proteins in a nonnative state. The folding funnel is a specific concept of the energy landscape theory of protein folding, which assumes that a protein's native state corresponds to its free energy minimum. The above diagram sketches one possible explanation for how proteins fold into their native structures that is based on the physics concept of minimizing free energy. In this model, the unfolded protein had both high entropy and high free energy. The high entropy corresponds to there being a large number of possible conformational states—the molecule can take on many different three-dimensional shapes. The high free energy means that the molecule is unstable, and flops easily between the different conformational states. As the protein starts to fold, the free energy drops and the number of available conformational states (denoted by the width of the funnel) decreases. There are local minima along the way that can trap the protein in a metastable state for some time, slowing its progress towards the free energy minimum. One such trap is indicated by the dashed red arrows. At the bottom of the funnel, the free energy is at a minimum and there is only one conformational state available to the protein molecule, called the 'native state'.
- Figure 1. Model for Folding of Newly Synthesized Proteins in the Prokaryotic Cytosol Ribosome-bound TF associates with emerging polypeptides and may transiently migrate with them. Subsequent folding of newly synthesized proteins occurs through different pathways (estimates of total protein molecules in %). Many proteins may fold without assistance by chaperones or with assistance by unknown factors. Specific subpopulations of substrates fold through assistance by either DnaK (K) with its DnaJ (J) and GrpE (E) cochaperones (co- and posttranslationally) or GroEL (EL) with its GroES (ES) cochaperone (posttranslationally). A small subset of these populations may re-quire transfer between these two chaperone systems through a free folding intermediate to reach the native state. nascent chain–associated complex (NAC) Figure 2. Model for Folding of Newly Synthesized Proteins in the Eukaryotic Cytosol Ribosome-bound NAC and in yeast the Hsp70 Ssb associate with emerging polypeptides and, similar to TF, may transiently migrate with them. Some proteins may fold without assistance or with assis-tance by unknown factors. A subpopulation requires co- and post-translational folding assistance by Hsp70 and Hsp40 (DnaJ) cochap-erones. Other proteins, such as actin and tubulin require prefoldin and CCT for folding. Crossover between pathways may occur with Hsp70/Hsp40 passing newly synthesized substrates to CTT (shown) or Hsp90 (not shown).
- The folding cycle for the green substrate starts with the association to the open ring (cis ring, I). After binding of ATP (II) the substrate is enclosed in a ™folding chamber∫ by the association of the GroES co-chaperone, which also consists of seven subunits and functions as a lid (III). The substrate is thereby prevented from interacting with proteins in solution, analogously to an infinite dilution. ATP and GroES binding and subsequent cooperative ATP hydrolysis in all GroEL subunits of the cis ring induce conformational changes in the cis ring leading to an enlargement of the folding chamber whereby the substrate binding sites of GroEL (red circles in Figure 5B) move away from the substrate (II, III, IV). This movement was shown to cause a global unfolding of the bound polypeptide.[27] It is assumed that unproductive hydrophobic interactions are thereby broken and the protein gets a new chance to fold correctly. Consequently, the protein substrate is lifted out of a local energy minimum of the folding funnel (see Figure 1). Binding of ATP, GroES, and substrate to the subunits of the idle ring (trans ring) induces the dissociation of the GroES co-chaperone of the cis ring and the release of the substrate (IV, V, VI). Some proteins fold while still enclosed in the GroEL folding chamber (Figure 5B, green substrate), others only after their release from GroEL (Figure 5B, blue substrate). Many proteins need several binding and release cycles before they reach their native structure.
- In the ATP state substrates associate to and dissociate from DnaK with high rates but affinity of DnaK to substrates is low. In the ADP state DnaK binds substrates with high affinity but substrate exchange rates are low. The DnaJ co-chaperone, which by itself also interacts with substrates through hydrophobic interactions, stimulates the ATPase activity of DnaK. Experimental evidence suggests that DnaJ ™recognizes∫ the substrate and passes it over to DnaK whereby ATP is hydrolyzed and a segment of the substrate is tightly enclosed…. Under physiological conditions substrate dissociation is controlled by ADP/ATP exchange that, in turn, is stimulated by the GrpE co-chaperone.