10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction BCB 444/544 Lecture 22  Secondary Structure Prediction  Tertiary Structure Prediction #22_Oct10

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Mon Oct 8 - Lecture 20 Protein Secondary Structure Prediction Chp 14 - pp Wed Oct 10 - Lecture 21 Protein Tertiary Structure Prediction Chp 15 - pp Thurs Oct 11 & Fri Oct 12 - Lab 7 & Lecture 22 Protein Tertiary Structure Prediction Chp 15 - pp Required Reading (before lecture)

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Assignments & Announcements ALL: HomeWork #3 √Due: Mon Oct 8 by 5 PM HW544: HW544Extra #1 √Due: Task Mon Oct 1 by noon Due: Task 1.2 & Task 2 - Fri Oct 12 by 5 PM 444 "Project-instead-of-Final" students should also submit: HW544Extra #1 √Due: Task Mon Oct 8 by noon Due: Task Fri Oct 12 by 5 PM

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction New Reading & Homework Assignment ALL: HomeWork #4 (posted online today) Due: Fri Oct 19 by 5 PM (one week from today) Read: Ginalski et al.(2005) Practical Lessons from Protein Structure Prediction, Nucleic Acids Res. 33: (PDF posted on website) Although somewhat dated, this paper provides a nice overview of protein structure prediction methods and evaluation of predicted structures. Your assignment is to write a summary of this paper - for details see HW#4 posted online & sent by on Fri Oct 12

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Seminars this Week - (yesterday) BCB List of URLs for Seminars related to Bioinformatics: Oct 11 Thurs Dr. Klaus Schulten (Univ of Illinois) - Baker Center Seminar The Computational Microscope 2:10 PM in E164 Lagomarcino Klaus_Schulten_Seminar.pdf Klaus_Schulten_Seminar.pdf Dr. Dan Gusfield (UC Davis) - Computer Science Colloquium ReCombinatorics: Combinatorial Algorithms for Studying History of Recombination in Populations 3:30 PM in Howe Hall Auditorium

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Seminars this Week - Fri (today) BCB List of URLs for Seminars related to Bioinformatics: Oct 12 Fri Dr. Edward Yu (Physics/BBMB, ISU) - BCB Faculty Seminar TBA: "Structural Biology" (see URL below) 2:10 PM in 102 Sci wsfilefield_abstract/Dr.-Ed-Yu.pdf wsfilefield_abstract/Dr.-Ed-Yu.pdf Dr. Srinivas Aluru (ECprE, ISU) - GDCB Seminar Consensus Genetic Maps: A Graph Theoretic Approach 4:10 PM in 1414 MBB newsfilefield_abstract/Dr.-Srinivas-Aluru.pdf

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Chp 12 - Protein Structure Basics SECTION V STRUCTURAL BIOINFORMATICS Xiong: Chp 12 Protein Structure Basics Amino Acids Peptide Bond Formation Dihedral Angles Hierarchy Secondary Structures Tertiary Structures Determination of Protein 3-Dimensional Structure Protein Structure DataBank (PDB)

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Experimental Determination of 3D Structure 2 Major Methods to obtain high-resolution structures 1.X-ray Crystallography (most PDB structures) 2.Nuclear Magnetic Resonance (NMR) Spectroscopy Note Advantages & Limitations of each method (See your lecture notes & textbook) For more info: Other methods (usually lower resolution, at present): Electron Paramagnetic Resonance (EPR - also called ESR, EMR) Electron microscopy (EM) Cryo-EM Scanning Probe Microscopies (AFM - Atomic Force Microscopy) Circular Dichroism (CD), several other spectroscopic methods

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction "Best" Resolution of Protein Structures High-resolution methods X-ray crystallography (< 1A  ) NMR (~ A  ) Lower-resolution methods Cryo-EM (~10-15A  ) Theoretical Models? Usually low resolution, at present, but Highly variable - & a few ~crystal data Baker & Sali (2000) Pevsner Fig 9.36

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Chp 13 - Protein Structure Visualization, Comparison & Classification SECTION V STRUCTURAL BIOINFORMATICS Xiong: Chp 13 Protein Structure Visualization, Comparison & Classification Protein Structural Visualization Protein Structure Comparison - later Protein Structure Classification

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Protein Structure Classification SCOP = Structural Classification of Proteins Levels reflect both evolutionary and structural relationships CATH = Classification by Class, Architecture,Topology & Homology DALI - (recently moved to EBI & reorganized) DALI Database (fold classification) Each method has strengths & weaknesses….

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Chp 14 - Secondary Structure Prediction SECTION V STRUCTURAL BIOINFORMATICS Xiong: Chp 14 Protein Secondary Structure Prediction Secondary Structure Prediction for Globular Proteins Secondary Structure Prediction for Transmembrane Proteins Coiled-Coil Prediction

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Secondary Structure Prediction Has become highly accurate in recent years (>85%) Usually 3 (or 4) state predictions: H =  -helix E =  -strand C = coil (or loop) (T = turn)

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Secondary Structure Prediction Methods 1st Generation methods Ab initio - used relatively small dataset of structures available Chou-Fasman - based on amino acid propensities (3-state) GOR - also propensity-based (4-state) 2nd Generation methods based on much larger datasets of structures now available GOR II, III, IV, SOPM, GOR V, FDM 3rd Generation methods Homology-based & Neural network based PHD, PSIPRED, SSPRO, PROF, HMMSTR, CDM Meta-Servers combine several different methods Consensus & Ensemble based JPRED, PredictProtein, Proteus

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Secondary Structure Prediction Servers Prediction Evaluation? Q 3 score - % of residues correctly predicted (3-state) in cross-validation experiments Best results? Meta-servers (scroll for 2' structure prediction) JPred PredictProtein Rost, Columbiahttp:// Best "individual" programs? ?? CDM Sen…Jernigan, ISUhttp://gor.bb.iastate.edu/cdm/ FDM(not available separately as server) Cheng…Jernigan, ISU GOR V Kloczkowsky…Jernigan, ISUhttp://gor.bb.iastate.edu/

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Developed by Jernigan Group at ISU Basic premise: combination of 2 complementary methods can enhance performance by harnessing distinct advantages of both methods; combines FDM & GOR V: FDM - Fragment Data Mining - exploits availability of sequence- similar fragments in the PDB, which can lead to highly accurate prediction - much better than GOR V - for such fragments, but such fragments are not available for many cases GOR V - Garnier, Osguthorpe, Robson V - predicts secondary structure of less similar fragments with good performance; these are protein fragments for which FDM method cannot find suitable structures For references & additional details: Consensus Data Mining (CDM)

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Where Find "Actual" Secondary Structure? In the PDB

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction How Does Predicted Secondary Structure Compare? e.g., from CMD QueryMAATAAEAVASGSGEPREEAGALGPAWDESQLRSYSFPTRPIPRLSQSDPRAEELIENEE GOR VCCCCHHHHHHHHCCHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHCCCC FDMCCCCCCCCCCCCCCCCCEECCCCCCCCCHHHCCCCCCEECCCCCCCCCCHHHHHHHHCCC CDMCCCCHHHHHHCCCCCCCEECCCCCCCCCHHHCCCCCCEECCCCCCCCCCHHHHHHHHCCC DSSP Author

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Secondary Structure Prediction: for Different Types of Proteins/Domains For Complete proteins: Globular Proteins - use methods previously described Transmembrane (TM) Proteins - use special methods (next slides) For Structural Domains: many under development: Coiled-Coil Domains (Protein interaction domains) Zinc Finger Domains (DNA binding domains), others…

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction SS Prediction for Transmembrane Proteins Transmembrane (TM) Proteins Only a few in the PDB - but ~ 30% of cellular proteins are membrane-associated ! Hard to determine experimentally, so prediction important TM domains are relatively 'easy' to predict! Why? constraints due to hydrophobic environment 2 main classes of TM proteins:  - helical  - barrel

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction SS Prediction for TM  -Helices  - Helical TM domains: Helices are amino acids long (span the membrane) Predominantly hydrophobic residues Helices oriented perpendicular to membrane Orientation can be predicted using "positive inside" rule Residues at cytosolic (inside or cytoplasmic) side of TM helix, near hydrophobic anchor are more positively charged than those on lumenal (inside an organelle in eukaryotes) or periplasmic side (space between inner & outer membrane in gram-negative bacteria) Alternating polar & hydrophobic residues provide clues to interactions among helices within membrane Servers? TMHMM or HMMTOP - 70% accuracy - confused by hydrophobic signal peptides ( short hydrophobic sequences that target proteins to the endoplasmic reticulum, ER) Phobius - 94% accuracy - uses distinct HMM models for TM helices & signal peptide sequences

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction SS Prediction for TM  -Helices  - Helical TM domains: Helices are amino acids long (span the membrane) Predominantly hydrophobic residues Helices oriented perpendicular to membrane Orientation can be predicted using "positive inside" rule Residues at cytosolic (inside or cytoplasmic) side of TM helix, near hydrophobic anchor are more positively charged than those on lumenal (inside an organelle in eukaryotes) or periplasmic side (space between inner & outer membrane in gram-negative bacteria) Alternating polar & hydrophobic residues provide clues to interactions among helices within membrane Servers? TMHMM or HMMTOP - 70% accuracy - confused by hydrophobic signal peptides ( short hydrophobic sequences that target proteins to the endoplasmic reticulum, ER) Phobius - 94% accuracy - uses distinct HMM models for TM helices & signal peptide sequences

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction SS Prediction for TM  - Barrels   - Barrel TM domains:   -strands are amphipathic (partly hydrophobic, partly hydrophilic) Strands are amino acids long Every 2nd residue is hydrophobic, facing lipid bilayer Other residues are hydrophilic, facing "pore" or opening Servers? Harder problem, fewer servers… TBBPred - uses NN or SVM (more on these ML methods later) Accuracy ?

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Prediction of Coiled-Coil Domains Coiled-coils S uperhelical protein motifs or domains, with two or more interacting  -helices that form a "bundle" Often mediate inter-protein (& intra-protein) interactions 'Easy' to detect in primary sequence: Internal repeat of 7 residues (heptad) 1 & 4 = hydrophobic (facing helical interface) 2,3,5,6,7 = hydrophilic (exposed to solvent) Helical wheel representation - can be used manually detect these, based on amino acid sequence Servers? Coils, Multicoil - probability-based methods 2Zip - for Leucine zippers = special type of CC in TFs: characterized by Leu-rich motif: L-X(6)-L-X(6)-L-X(6)-L

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Chp 15 - Tertiary Structure Prediction SECTION V STRUCTURAL BIOINFORMATICS Xiong: Chp 15 Protein Tertiary Structure Prediction Methods Homology Modeling Threading and Fold Recognition Ab Initio Protein Structural Prediction CASP

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Structural Genomics - Status & Goal ~ 20,000 "traditional" genes in human genome (recall, this is fewer than earlier estimate of 30,000) ~ 2,000 proteins in a typical cell > 4.9 million sequences in UniProt (Oct 2007) > 46,000 protein structures in the PDB (Oct 2007) Experimental determination of protein structure lags far behind sequence determination! Goal: Determine structures of "all" protein folds in nature, using combination of experimental structure determination methods (X-ray crystallography, NMR, mass spectrometry) & structure prediction

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Structural Genomics Projects TargetDB: database of structural genomics targets

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Protein Sequence & Structure: Analysis Diamond STING Millennium - Many useful structure analysis tools, including Protein Dossier SwissProt (UniProt) Protein knowledgebase InterPro S equence analysis tools

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Protein Structure Prediction or Protein Folding Problem "Major unsolved problem in molecular biology" In cells:spontaneous assisted by enzymes assisted by chaperones In vitro: many proteins can fold to their "native" states spontaneously & without assistance but, many do not!

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Deciphering the Protein Folding Code Protein Structure Prediction or "Protein Folding" Problem Given the amino acid sequence of a protein, predict its 3-dimensional structure (fold) "Inverse Folding" Problem Given a protein fold, identify every amino acid sequence that can adopt that 3-dimensional structure

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Protein Structure Prediction Structure is largely determined by sequence BUT: Similar sequences can assume different structures Dissimilar sequences can assume similar structures Many proteins are multi-functional 2 Major Protein Folding Problems: 1- Determination of folding pathway 2- Prediction of tertiary structure from sequence Both still largely unsolved problems

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Steps in Protein Folding 1-"Collapse"- driving force is burial of hydrophobic aa’s (fast - msecs) 2- Molten globule - helices & sheets form, but "loose" (slow - secs) 3- "Final" native folded state - compaction & rearrangement of some 2' structures Native state? - assumed to be lowest free energy - may be an ensemble of structures

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Protein Dynamics Protein in native state is NOT static Function of many proteins requires conformational changes, sometimes large, sometimes small Globular proteins are inherently "unstable" (NOT evolved for maximum stability) Energy difference between native and denatured state is very small (5-15 kcal/mol) (this is equivalent to ~ 2 H-bonds!) Folding involves changes in both entropy & enthalpy

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction Difficulty of Tertiary Structure Prediction Folding or tertiary structure prediction problem can be formulated as a search for minimum energy conformation Search space is defined by psi/phi angles of backbone and side-chain rotamers Search space is enormous even for small proteins! Number of local minima increases exponentially with number of residues Computationally it is an exceedingly difficult problem!

10/12/07BCB 444/544 F07 ISU Dobbs #22 - Secondary & Tertiary Structure Prediction From Thursday's Lab: Homology Modeling - using SWISS-MODEL Threading - using 3-D JURY (BioinfoBank, a METAserver) Be sure to take a look at CASP contest: CASP7 contest in