Transcript Lecture_11_2005

Structural proteomics
Handouts. Proteomics section from
book already assigned.
What is structural
proteomics/genomics?
• High-throughput determination of the 3D structure
of proteins
• Goal: to be able to determine or predict the
structure of every protein.
– Direct determination - X-ray crystallography and
nuclear magentic resonance (NMR).
– Prediction
• Comparative modeling • Threading/Fold recognition
• Ab initio
Why structural proteomics?
• To study proteins in their active
conformation.
– Study protein:drug interactions
– Protein engineering
• Proteins that show little or no similarity at
the primary sequence level can have
strikingly similar structures.
An example
• FtsZ - protein required for cell division in
prokaryotes, mitochondria, and chloroplasts.
• Tubulin - structural component of microtubules important for intracellular trafficking and cell
division.
• FtsZ and Tubulin have limited sequence similarity
and would not be identified as homologous
proteins by sequence analysis.
FtsZ and tubulin have little
similarity at the amino acid
sequence level
Burns, R., Nature 391:121-123
Picture from E. Nogales
Are FtsZ and tubulin
homologous?
• Yes! Proteins that have conserved secondary
structure can be derived from a common
ancestor even if the primary sequence has
diverged to the point that no similarity is
detected.
Current state of structural
proteomics
• As of Feb. 2002 - 16,500 structures
– Only 1600 non-redundant structures
• To identify all possible folds - predicted another
16,000 novel sequences needed for 90% coverage.
– Of the 2300 structures deposited in 2000, only 11%
contained previously unidentified folds.
• Overall goal - directly solve enough structures
directly to be able to computationally model all
future proteins.
Protein domains - structure
• “clearly recognizable portion of a protein
that folds into a defined structure”
– Doesn’t have to be the same as the domains we
have been investigating with CDD.
– RbsB proteins as an example.
Main secondary structure
elements
a-helix - right handed helical structure
b-sheet - composed of two or more b-strands,
conformation is more “zig-zag” than
helical.
Images from http://www-structure.llnl.gov/Xray/tutorial/protein_structure.htm
http://www.expasy.org/swissmod/course/text/chapter1.htm
Folds/motifs - tertiary structure
• How these secondary structure elements
come together to form structure.
– Helix-turn-helix
• Determining the structure of nearly all folds
is the goal of structural biology
Quaternary structure
• Refers to the structure formed by more than
one polypeptide.
• Many proteins function as complexes - best
to know the structure of the complex rather
than each individual
– Proteins may have different conformations
when in a complex vs. alone.
X-Ray Crystallography
• Make crystals of your protein
– 0.3-1.0mm in size
– Proteins must be in an ordered, repeating
pattern.
• X-ray beam is aimed at crystal and data is
collected.
• Structure is determined from the diffraction
data.
Image from http://www-structure.llnl.gov/Xray/101index.html
Schmid, M. Trends in Microbiolgy, 10:s27-s31.
X-ray crystallography
• Protein must crystallize.
– Need large amounts (good expression)
– Soluble (many proteins aren’t, membrane proteins).
• Need to have access to an X-ray beam.
• Solving the structure is computationally intensive.
• Time - can take several months to years to solve a
structure
– Efforts to shorten this time are underway to make this
technique high-throughput.
Nuclear Magnetic Resonance
Spectroscopy (NMR)
• Can perform in solution.
– No need for crystallization
• Can only analyze proteins that are <300aa.
– Many proteins are much larger.
– Can’t analyze multi-subunit complexes
• Proteins must be stable.
Structure modeling
• Comparative modeling
– Modeling the structure of a protein that has a high
degree of sequence identity with a protein of known
structure
– Must be >30% identity to have reliable structure
• Threading/fold recognition
– Uses known fold structures to predict folds in primary
sequence.
• Ab initio
– Predicting structure from primary sequence data
– Usually not as robust, computationally intensive
Structure of the ribosome
• Ribosome - made up of 3 RNA molecules
and over 50 proteins.
• Structure of the 70S ribosome solved by
combining several models of the individual
30S and 50S subunits
Bacterial ribosome
• Ribosome is a 2.3 MDa complex
– 50S and 30S subunits
– 54 proteins and 3 RNAs (23S, 16S,
and 5S RNAs)
– Can account for ~50% of cell mass
during rapid growth.
– Major target for many antibiotics.
• Ribosome is a ribozyme!
Ramakrishnan (2002) Cell. 108:557-572