Download file (4.1 MB, ppt)

Protein structure and function
Part III
Marie-Véronique CLEMENT
Associate Professor
Yong Loo Lin School of Medicine
NUS Graduate School for Integrative Science and Engineering
Department of Biochemistry
National University of Singapore
8 Medical Drive, MD 7 #03-15
Singapore 117597
Tel: (65) 68747985
Fax: (65) 67791453
E-mail: [email protected]
Structure of the hemaglutinine
protein
(a long multimeric molecule whose three identical subunits are each composed of two chains, HA1
and HA2).
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Primary structure
(1 letter code used)
b strands
random
coils
a helices
Secondary structure
Protein
domains
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Tertiary
structure
Quaternary
structure
Protein domains:
Any part of a protein that can fold
independently into a compact, stable
structure. A domain usually contains
between 40 and 350 amino acids.
•
• A domain is the modular unit from which
many larger proteins are constructed.
• The different domain of protein are often
associated with different functions.
Protein domains
Cytochrome b562
A single domain protein
involved in electron transport
in mitochondria
The NAD-binding
domain of
the enzyme lactic
dehydrogenase
The variable domain
of an immunoglobulin
The Src protein
The modular nature of proteins:
EGF domain
Immunoglobulin domain
Membrane spanning domain
(tissue plasminogen activator)
Fibronectine
domain
Chymotryptic domain
Epidermal growth factor: (EGF) is generated by proteolytic cleavage of a precursor protein
containing multiple EGF domains (orange).
The EGF domain also occurs in Neu protein and in tissue plasminogen activator (TPA).
Other domains, or modules, in these proteins include a chymotryptic domain (purple), an
immunoglobulin domain (green), a fibronectin domain (yellow), a membrane-spanning domain
(pink), and a kringle domain (blue).
[Adapted from I. D. Campbell and P. Bork, 1993, Curr. Opin. Struc. Biol. 3:385.]
How protein structures are determined?
The majority of protein structures known to date have been solved with
the experimental technique of
• X-ray crystallography,
which typically provides data of high resolution but provides no timedependent information on the protein's conformational flexibility.
• NMR (nuclear magnetic resonance spectroscopy),
which provides somewhat lower-resolution data in general and is limited
to relatively small proteins, but can provide time-dependent information
about the motion of a protein in solution.
More is known about the tertiary structural features of soluble globular
proteins than about membrane proteins because the latter class is
extremely difficult to study using these methods.
An X-ray diffraction image for the protein myoglobin.
The first protein crystal structure was of sperm whale myoglobin, as
determined by Max Perutz and Sir John Cowdery Kendrew in 1958,
which led to a Nobel Prize in Chemistry.
The X-ray diffraction analysis of myoglobin was originally motivated by
the observation of myoglobin crystals in dried pools of blood on the decks
of whaling ships.
NMR is a field of structural biology, that applies nuclear magnetic
resonance spectroscopy to investigating proteins
The field was pioneered by among others, Kurt Wüthrich, who won the Nobel prize in 2002,
Pacific Northwest National Laboratory's high magnetic field
(800 MHz) NMR spectrometer being loaded with a sample.
The NMR sample is prepared
in a thin walled glass tube.
Protein NMR is performed on aqueous samples of highly purified protein.
Sample consist of between 300 and 600 microlitres with a protein concentration in the range 0.1 – 3
millimoles.
The source of the protein can be either natural or produced in an expression system using
recombinant DNA techniques through genetic engineering.
Function of
peptides and proteins
Oxytocin and vasopressin are two peptide hormones with
very similar structure, but with very different biological
activities.
Interestingly, their structures only differ by one amino acid
residue (the hydrophobic LEU number 8 in oxytocin is replaced
by a hydrophilic ARG residue in vasopressin).
Oxytocin is a potent stimulator of uterine smooth muscle, and
also stimulates lactation.
Read Table 3.2 in Devlin
for other examples of
biologically active peptides
Vasopressin, also know as antidiuretic hormone (ADH), has
no effect on uterine smooth muscle, but causes reabsorbtion
of water by the kidney, thus increasing blood pressure.
Function of proteins
• Enzymatic catalysis
• Transport and storage (the protein hemoglobin,
albumins)
• Coordinated motion (actin and myosin).
Proteins
are the most
important buffers in
the body.
• Mechanical support (collagen).
• Immune protection (antibodies)
• Generation and transmission of nerve impulses
- some amino acids act as neurotransmitters,
receptors for neurotransmitters, drugs, etc. are
protein in nature. (the acetylcholine receptor),
• Control of growth and differentiation transcription factors
Hormones
growth factors ( insulin or thyroid stimulating
hormone)
Why?
(a)
Protein molecules
possess basic and
acidic groups which act
as H+ acceptors or
donors respectively if
H+ is added or removed.
(a)
Proteins are the most important buffers in the body. They are mainly
intracellular and include haemoglobin.
(b) The plasma proteins are buffers but the absolute amount is small compared
to intracellular protein.
(c)
Protein molecules possess basic and acidic groups which act as H+
acceptors or donors respectively if H+ is added or removed.
• Many proteins (thousands!) present in blood plasma
• Proteins contain weakly acidic (glutamate, aspartate) and basic
(lysine, arginine, histidine) side chains (or R groups)
• At neutral pH, only histidine residues (containing imidazole R
group with pKa ~ 6.0) in proteins can act as a buffer component
• Haemoglobin with 38 histidine/tetramer is a good buffer
• N-terminal groups of proteins (pKa ~ 8.0) can also act as a
buffer component
Proteins play crucial roles in almost every biological process. They are
responsible in one form or another for a variety of physiological functions
including
Enzymatic catalysis
Transport and storage
Coordinated motion
Mechanical support
Immune protection
Generation and transmission of nerve impulses
Control of growth and differentiation
Enzymatic catalysis almost all biological reactions are enzyme catalyzed.
Enzymes are known to increase the rate of a biological reaction by a factor of 10 to the 6th power!
There are several thousand enzymes which have been identified to date.
Transport and storage - small molecules are often carried by proteins in the physiological setting
(for example, the protein hemoglobin is responsible for the transport of oxygen to tissues). Many drug
molecules are partially bound to serum albumins in the plasma.
The binding of oxygen is affected by molecules such as carbon
monoxide (CO) (for example from tobacco smoking, cars and
furnaces).
CO competes with oxygen at the heme binding site. Hemoglobin
binding affinity for CO is 200 times greater than its affinity for
oxygen, meaning that small amounts of CO dramatically reduces
hemoglobin's ability to transport oxygen. When hemoglobin
combines with CO, it forms a very bright red compound called
carboxyhemoglobin.
3-dimensional structure of hemoglobin.
The four subunits are shown in red and
yellow, and the heme groups in green.
When inspired air contains CO levels as low as 0.02%, headache
and nausea occur; if the CO concentration is increased to 0.1%,
unconsciousness will follow. In heavy smokers, up to 20% of the
oxygen-active sites can be blocked by CO.
Coordinated motion - muscle is mostly protein, and muscle contraction is mediated by the sliding
motion of two protein filaments, actin and myosin.
Platelet activation is a controlled
sequence of actin filament:
Severing
Uncapping
Elongating
Cross linking
That creates a dramatic shape change
in the platelet
Platelet before activation
Activated platelet
Activated platelet
at a later stage than C)
Mechanical support - skin and bone are strengthened by the protein collagen.
Abnormal collagen synthesis or
structure causes dysfunction of
• cardiovascular organs,
• bone,
• skin,
• joints
• eyes
Refer to Devlin
Clinical correlation 3.4 p121
Immune protection - antibodies are protein structures that are responsible for reacting with specific
foreign substances in the body.
Generation and transmission of nerve impulses Some amino acids act as neurotransmitters, which transmit electrical signals from one nerve cell to another. In
addition, receptors for neurotransmitters, drugs, etc. are protein in nature.
An example of this is the acetylcholine receptor, which is a protein structure that is embedded in postsynaptic
neurons.
GABA:
gamma Amino butyric acid
Synthesised from glutamate
GABA acts at inhibitory synapses in the
brain. GABA acts by binding to specific
receptors in the plasma membrane of both
pre- and postsynaptic neurons.
Neurotransmetter
Control of growth and differentiation proteins can be critical to the control of growth, cell differentiation and expression of DNA.
For example, repressor proteins may bind to specific segments of DNA, preventing expression and thus the
formation of the product of that DNA segment.
Also, many hormones and growth factors that regulate cell function, such as insulin or thyroid stimulating hormone
are proteins.
Insuline
Membrane transport proteins
STRUCTURE - FUNCTION
RELATIONSHIPS
In general, all globular proteins have
distinctive
3D structures that are
specialized for their particular functions.
Shape and function
The relationship between shape and function of proteins:
The relationship between shape and function of proteins:
Protein degradation:
Disease and protein folding:
Disease
Exemple:
Neurodegenerative
diseases
HOT Areas of Medical Research
Human Genome
sequencing is completed
Application in Biology and
Medicine just beginning
e.g., Cloning of a disease gene is
the first step in understanding
the basic defects and rational
treatment
Structural and functional
characterization of all novel
PROTEINS will unravel new
disease genes.
Shape and function
In globular proteins, tertiary interactions are frequently stabilized by the sequestration of
hydrophobic amino acid residues in the protein core, from which water is excluded, and by the
consequent enrichment of charged or hydrophilic residues on the protein's water-exposed surface.
In secreted proteins that do not spend time in the cytoplasm, disulfide bonds between cysteine
residues help to maintain the protein's tertiary structure.
A variety of common and stable tertiary structures appear in a large number of proteins that are
unrelated in both function and evolution - for example, many proteins are shaped like a TIM barrel,
named for the enzyme triosephosphateisomerase.
Another common structure is a highly stable dimeric coiled-coil structure composed of four alpha
helices.
Proteins are classified by the folds they represent in databases like SCOP and CATH.