Download Protein Structure & Function PPT

11/4/05
Protein Structure & Function
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
1
Announcements
Exam 2 - Has been graded
- Will be returned at end of class today
Grade statistics – 444 Average = 81/100
544 Average = 100/118
Questions?
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
2
Announcements
BCB 544 Projects - Important Dates:
Nov 2 Wed noon - Project proposals due to David/Drena
Nov 4 Fri PM - Approvals/responses & tentative
presentation schedule to students
Dec 2 Fri noon - Written project reports due
Dec 5,7,8,9 class/lab
- Oral Presentations (20')
(Dec 15 Thurs = Final Exam)
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
3
Bioinformatics Seminars
Nov 4 Fri 12:10 PM BCB Faculty Seminar in E164 Lago
How to do sequence alignments on parallel computers
Srinivas Aluru, ECprE & Chair, BCB Program
http://www.bcb.iastate.edu/courses/BCB691F2005.html
Next week:
Nov 10 Thurs 3:40 PM ComS Seminar in 223 Atanasoff
Computational Epidemiology
Armin R. Mikler, Univ. North Texas
http://www.cs.iastate.edu/~colloq/#t3
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
4
Bioinformatics Seminars
CORRECTION:
Week after next - Baker Center/BCB Seminars:
(seminar abstracts available at above link)
Nov 14 Mon 1:10 PM Doug Brutlag, Stanford
Discovering transcription factor binding sites
Nov 15 Tues 1:10 PM
Ilya Vakser, Univ Kansas
Modeling protein-protein interactions
both seminars will be in Howe Hall Auditorium
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
5
RNA Structure & Function/Prediction
Protein Structure & Function
Mon
Review - promoter prediction
Wed
RNA structure & function
RNA structure prediction
2' & 3' structure prediction
miRNA & target prediction - Lab 10
Fri
- a few more words re: Algorithms
Protein structure & function
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
6
Reading Assignment (for Fri/Mon)
Mount Bioinformatics
• Chp 10 Protein classification & structure prediction
http://www.bioinformaticsonline.org/ch/ch10/index.html
• pp. 409-491
• Ck Errata: http://www.bioinformaticsonline.org/help/errata2.html
Other? That should be plenty…
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
7
Review last lecture:
RNA Structure Prediction
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
8
miRNA and RNAi pathways
microRNA pathway
RNAi pathway
MicroRNA primary transcript
Exogenous dsRNA, transposon,
etc.
Drosha
precursor
Dicer
Dicer
siRNAs
miRNA
target mRNA
RISC
RISC
“translational repression”
and/or mRNA degradation
C Burge 2005
11/04/05
RISC
mRNA cleavage, degradation
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
9
miRNA Challenges for Computational Biology
• Find the genes encoding microRNAs
• Predict their regulatory targets
Computational Prediction of MicroRNA Genes & Targets
• Integrate miRNAs into gene regulatory pathways &
networks
Need to modify traditional paradigm of
"transcriptional control" primarily by protein-DNA
interactions to include miRNA regulatory mechanisms!
C Burge 2005
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
10
RNA structure prediction strategies
Secondary structure prediction
1) Energy minimization
(thermodynamics)
2) Comparative sequence analysis
(co-variation)
3) Combined experimental & computational
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
11
Secondary structure prediction strategies
1) Energy minimization (thermodynamics)
• Algorithm:
Dynamic programming to find
high probability pairs
(also, some genetic algorithms)
• Software:
Mfold - Zuker
Vienna RNA Package - Hofacker
RNAstructure - Mathews
Sfold - Ding & Lawrence
R Knight 2005
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
12
Secondary structure prediction strategies
2) Comparative sequence analysis (co-variation)
• Algorithms:
Mutual information
Stochastic context-free grammars
• Software:
ConStruct
Alifold
Pfold
FOLDALIGN
Dynalign
R Knight 2005
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
13
Secondary structure prediction strategies
3) Combined experimental & computational
• Experiment:
Map single-stranded vs double-stranded
regions in folded RNA
• How?
Enzymes: S1 nuclease, T1 RNase
Chemicals: kethoxal, DMS
R Knight 2005
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
14
Experimental RNA structure determination?
• X-ray crystallography
• NMR spectroscopy
• Enzymatic/chemical mapping
• Molecular genetic analyses
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
15
1) Energy minimization method
What are the assumptions?
Native tertiary structure or "fold" of an RNA
molecule is (one of) its "lowest" free energy
configuration(s)
Gibbs free energy = G in kcal/mol at 37C
= equilibrium stability of structure
lower values (negative) are more favorable
Is this assumption valid?
in vivo? - this may not hold, but we don't really know
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
16
Free energy minimization
What are the rules?
A
A
U
U
Basepair
A=U
A=U
What gives here?
G = -1.2 kcal/mole
A
U
U
A
Basepair
A=U
U=A
G = -1.6 kcal/mole
C Staben 2005
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
17
Energy minimization calculations:
Base-stacking is critical
AA
UU
-1.2
CG
GC
-3.0
AU or UA
UA
AU
-1.6
GC
CG
-4.3
AG, AC, CA, GA
UC, UG, GU, CU
-2.1
GU
UG
-0.3
CC
GG
-4.8
XG, GX
YU, UY
0
- Tinocco et al.
C Staben 2005
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
18
Nearest-neighbor parameters
Most methods for free energy minimization
use nearest-neighbor parameters (derived from
experiment) for predicting stability of an RNA
secondary structure (in terms of G at 37C)
& most available software packages use
the same set of parameters:
Mathews, Sabina, Zuker & Turner, 1999
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
19
Energy minimization - calculations:
Total free energy of a specific
conformation for a specific RNA
molecule = sum of incremental
energy terms for:
• helical stacking
(sequence dependent)
• loop initiation
• unpaired stacking
(favorable "increments" are < 0)
Fig 6.3
Baxevanis &
Ouellette 2005
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
20
But how many possible conformations for a
single RNA molecule?
Huge number:
Zuker estimates (1.8)N possible secondary
structures for a sequence of N nucleotides
for 100 nts (small RNA…) =
3 X 1025 structures!
Solution? Not exhaustive enumeration…
 Dynamic programming
O(N3) in time
O(N2) in space/storage
iff pseudoknots excluded, otherwise:
O(N6 ), time
O(N4 ), space
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
21
Algorithms based on energy minimization
For outline of algorithm used in Mfold, including
description of dynamic programming recursion, please
visit Michael Zuker's lecture:
http://www.bioinfo.rpi.edu/~zukerm/lectures/RNAfold-html
From this site, you may also download his lecture as
either PDF or PS file.
Hmmm, something based on this might make an interesting "Final
Exam" question: how could one apply dynamic programming
approaches learned in first half of course to RNA structure
prediction problem?
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
22
2) Comparative sequence analysis
(co-variation)
Two basic approaches:
• Algorithms constrained by initial alignment
Much faster, but not as robust as unconstrained
Base-pairing probabilities determined by a
partition function
• Algorithms not constrained by initial alignment
Genetic algorithms often used for finding an
alignment & set of structures
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
23
RNA Secondary structure prediction:
Performance?
How evaluate?
• Not many experimentally determined structures
currently, ~ 50% are rRNA structures
so "Gold Standard" (in absence of tertiary structure):
compare with predicted RNA secondary
structure with that determined by comparative
sequence analysis (!!??) using Benchmark Datasets
NOTE: Base-pairs predicted by comparative sequence
analysis for large & small subunit rRNAs are 97% accurate
when compared with high resolution crystal structures!
- Gutell, Pace
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
24
RNA Secondary structure prediction:
Performance?
1) Energy minimization (via dynamic programming)
73% avg. prediction accuracy - single sequence
2) Comparative sequence analysis
97% avg. prediction accuracy - multiple sequences
(e.g., highly conserved rRNAs)
much lower if sequence conservation is lower &/or
fewer sequences are available for alignment
3) Combined - recent developments:
combine thermodynamics & co-variation
& experimental constraints? IMPROVED RESULTS
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
25
RNA structure prediction strategies
Tertiary structure prediction
Requires "craft" & significant user input & insight
1) Extensive comparative sequence analysis to predict
tertiary contacts (co-variation)
e.g., MANIP - Westhof
2) Use experimental data to constrain model building
e.g., MC-CYM - Major
3) Homology modeling using sequence alignment &
reference tertiary structure (not many of these!)
4) Low resolution molecular mechanics
e.g., yammp - Harvey
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
26
New Today:
Protein Structure & Function
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
27
Protein Structure & Function
Protein structure - primarily determined by sequence
Protein function - primarily determined by structure
• Globular proteins: compact hydrophobic core &
hydrophilic surface
• Membrane proteins: special hydrophobic surfaces
• Folded proteins are only marginally stable
• Some proteins do not assume a stable "fold" until they
bind to something = Intrinsically disordered
 Predicting protein structure and function can be very
hard -- & fun!
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
28
4 Basic Levels of Protein Structure
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
29
Primary & Secondary Structure
Primary
• Linear sequence of amino acids
• Description of covalent bonds linking aa’s
Secondary
• Local spatial arrangement of amino acids
• Description of short-range non-covalent
interactions
• Periodic structural patterns: -helix, b-sheet
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
30
Tertiary & Quaternary Structure
Tertiary
• Overall 3-D "fold" of a single polypeptide chain
• Spatial arrangement of 2’ structural elements;
packing of these into compact "domains"
• Description of long-range non-covalent interactions
(plus disulfide bonds)
Quaternary
• In proteins with > 1 polypeptide chain, spatial
arrangement of subunits
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
31
"Additional" Structural Levels
•
•
•
•
Super-secondary elements
Motifs
Domains
Foldons
11/04/05
D Dobbs ISU - BCB 444/544X: Protein Structure & Function
32