Download Chapter01 Introduction Amino Acids, Peptides and Proteins (绪论

教
案
~ 2007 学年 第一 学期
2006
学 院
教
名 称
研
生命科学学院
室
课 程
名 称
授 课
对 象
授 课
教 师
陈文利
称
副教授
职
教 材
名 称
2006 年 9 月
生物化学
2005 级生物技术专业
现代生物化学
日
授课题目（教学章、节或主题）
：
教学器材
与工具
Chapter1 Introduction Amino Acids, Peptides and
授课时间
Proteins (绪论 氨基酸，肽和蛋白质)
多媒体设施、黑板与
笔
第 1，2，3 周一第 1-8
节
教学目的、要求（例如识记、理解、简单应用、综合应用等层次）
：
介绍生物化学的进展，激发学生学习生物化学的兴趣，了解为什么要学习生物化学和
本课程的学习内容与安排。介绍氨基酸结构，蛋白质一级结构。重点介绍蛋白质二级
结构及主要性质等电点等。
教学内容（包括基本内容、重点、难点）
：
Introduction to Biochemistry
What is Biochemistry?
Bio + Chemistry= Biochemistry
The Goals of Biochemistry
Describe structure, organization, function of cells in molecular terms
(1) Structural Chemistry
(2) Metabolism
(3) Molecular Genetics
生物化学的内容主要有三方面 1．研究组成生物体的基本物质（糖类、脂类、蛋白质、核酸）以
及对体内的化学反应起催化和调节作用的生物活性物质(酶、维生素、激素)的结构、性质和功能。
2．研究糖类、脂类、蛋白质和核酸在生命活动过程中进行的化学变化，也就是新陈代谢，以及
代谢过程中能量的转换和代谢调节。 3．研究遗传信息的传递和表达。
A Tip on how to study Biochemistry well
Just do as a proverb says:
I hear, and I forget,
I see, and I remember,
I do, and I understand.
Chapter1 Amino Acids, Peptides and Proteins
Amino acid structure
Amino Acid Classification
The isoelectric point of an amino acid occurs at the pH where the amino acid exists as the
zwitterion.
pI (isoelectric point) = the pH at which the number of positive and negative charges on a
population of molecules is equal (i.e. no net charge).
pI (isoelectric point)：
No net charge
Minimum solubility in water
protein will precipitate out at its isoelectric point
can separate amino acids and peptides based in electrophoresis:
+ charged amino acids move to - electrode
- charged amino acids move to + electrode
amino acids at their isoelectric points do not move
Peptides & Proteins
Peptides contain 50 or fewer amino acids and are further classified below.
Dipeptides contain 2 amino acids.
Tripeptides contain 3 amino acids.
Polypeptides contain 50 or fewer amino acids.
Proteins contain greater than 50 amino acids.
An amino acid in the peptides or proteins is called amino acid residue
Biological Functions of Proteins
Proteins are the agents of biological function
Enzymes - Ribonuclease
Signal transduction – Insulin and its receptor
Control of Gene expression-Transcription factors
Immunity-Antibody
Transport and Storage - Hemoglobin
Structural proteins – Hair, Collagen
Contractile proteins - Actin, Myosin
Exotic proteins - Antifreeze proteins in fish
Hierarchy of protein structure
Primary Structure (1º) : Unique sequence of amino acids: sequence is determined by genetic
material
Secondary Structure (2º) : coiling /folding as a result of hydrogen bonding
Tertiary Structure (3º) : 3-D shape due to bonding of R- groups
Quaternary Structure (4º) : association of 2 or more polypeptides; Ex HGB ; not all have this
level
Dipeptides: two amino acids linked by peptide bond (amide linkage)
Peptides are written so that the free NH2 group is on the left and the free COOH group is on the
right.
N-terminus: alanine
C-terminus: aspartic acid
Ala-Asp
alanylaspartic acid
Secondary Structure
The Coplanar Nature of the Peptide Bond
Four patterns
–a (alpha) helix
– (beta) sheet
– turn
–Random coil
Six atoms of the peptide group lie in a plane!
Resonance explains partial double bond
Consequences of the Amide Plane
Only two degrees of freedom per residue for the peptide chain
Angle about the C-N bond is phi ()
Angle about the C -C bond is psi ()
The entire path of the peptide backbone is known if all phi and psi angles are specified
Some values of phi and psi are more likely than others.
The formation of the a-helix is spontaneous and is stabilized by Stabilised by H-bonds in backbone
NH…O=C spaced four residues apart.
The a-helix
Right-handed
Side-chains point out
Residues per turn: 3.6
Rise per residue: 1.5 Å
Rise per turn: 5.4 Å
 sheets
Also first postulated by Pauling and Corey, 1951
Strands may be parallel (0.325 nm between two residues) or antiparallel (0.347 nm between
two residues)
Nearly fully extended C=O, N-H groups form H-bonds between neighbouring strands
Sheets pleat to maintain correct H-bond stereochemistry
Side chains point alternatively on opposite sides of the sheet
The Beta Turn (tight turn, or -bend)
Beta turns connect beta strands and reverse the direction of beta strands;
Pro and gly have high propensity for beta turns;
The carbonyl oxygen of the ith residue forms H-bond with the amide proton of the (i+3)th
residue;
Tight turn promotes formation of antiparallel beta sheets.
Tertiary structure formed through side chain interactions
Tertiary
Complete three-dimensional structure
Native conformation: functional structure
Most stable conformation
Due to weak interactions between side (R) groups as well as covalent disulfide bonds
Weak interactions
Hydrogen bonds
Electrostatic interactions (ionic bonds)
Hydrophobic interactions
Van der Waals interactions
Quaternary: arrangement of subunits (in multisubunit protein)
Held together by weak interactions between side (R/functional) groups as well as covalent disulfide
bonds
Structure-function relationship
Function is derived from structure
Structure is derived from sequence
Similar sequences have similar functions
Similar function often implies evolutionary relatedness
Sequence similarity suggests common evolutionary origin
Many diseases are related to anomaly of some proteins
-Cystic fibrosis, sickle cell anemia and mad cow disease
Sickle-cell disease
Single specific amino acid change causes change in protein structure and solubility
Results in change in cell shape
Causes cells to clog blood vessels
Some Properties of Proteins
pI value and solubility
Denature and Re-nature
Denaturation
Disruption of the normal 3D structure of a protein, such that it loses biological activity.
Usually caused by heat, Chemicals or changes in pH.
Usually irreversible. A cooked egg cannot be “uncooked.”
Solubility of Proteins
Solubility of proteins are strongly influenced by solution pH and salt concentrations.
– pH dependence of solubility
– In general, solubility is least at pI
because of least electrostatic
interactions with solvent
– Salt dependence of solubility
Salting in refers to the phenomenon that
solubility increases as salt concentration
increases;
Salting out refers to the phenomenon
that solubility decreases as salt
concentration increases;
The classic protein fractionation method
called ammonium precipitation is based
on the salting out phenomenon.
Protein Isolation
Common methods used in protein isolation from cells or tissues:
– Size exclusion (Gel filtration);
– Affinity chromatography;
– Ion exchange chromatography;
– Hydrophobic Interaction chromatography;
Assessment of protein purity and size by SDS PAGE (Sodium dodecylsulfate polyacrylamide
gel electrophoresis)
Size Exclusion
Separation of proteins based on their size:
The beads are composed of dextran polymers (sephadex), agarose (Sepharose),
polyacrylamide (Sephacryl or BioGel P). Each bead contains pores of approximate
macromolecule sizes. Larger molecules will travel through less pores, thus migrate faster.
Smaller molecules will travel through more pores, thus migrate slower. The molecules
passively distribute between the volume outside the porous beads (V0) and the volume inside
the beads (Vi) dependent on their ability to enter the pores. If the total volume of the column is
Vt,
V t = Vo + Vi
Affinity Chromatography
Affinity chromatography makes use of the affinity between a ligand and the protein of interests.
Ligands are usually immobilized through covalent bonds on insoluble matrix, such as cellulose
or polyacrylamide. The protein of interests become bound to the matrix while other proteins
flow through the column. After washing the protein bound to the matrix can be eluted by adding
completing groups such as the free ligand, or reagents that disrupt the interactions. Examples
of ligand-protein interactions include those between antibodies and antigens, those between
Ni+ and poly-histidine tag.
Ion Exchange Chromatography
Ion exchange chromatography makes use of electrostatic properties of the protein of interests.
Charged polymers are usually immobilized through covalent bonds on insoluble matrix, such
as cellulose or polyacrylamide. The protein of opposite charge become bound to the matrix
while other proteins flow through the column. After washing, the protein bound to the matrix can
be eluted by adding salts.
Ion Exchange Chromatography
Electrostatic properties of a protein determine the type of ion exchange resins it interacts with.
In principle:
– Protein is positively charged if solution pH < pI; It should bind to negatively charged
resins, or cation exchanger;
– Protein is negatively charged if solution pH > pI; It should bind to positively charged
resins, or anion exchanger;
– Note that in practice, protein surface has local charges that may different from total
charge of the protein.
Examples of cation exchangers include : carboxymethyl (CM) and sulfopropyl (SP)
Examples of anion exchangers include: diethylaminoethyl (DEAE), quaternary amine (QAE)
Hydrophobic Interaction
Hydrophobic interaction chromatography makes use of the hydrophobic patches on protein and
their interactions with hydrophobic resins.
For instance, phenyl sepharose is a strong hydrophobic resin that is made by covalently attach
phenyl group to agarose supporting matrix. Proteins bind to phenyl group by virtue of
hydrophobic interactions. Such interactions are most favored in the presence of high salts.
Thus a typical procedure for running hydrophobic chromatography is to first bind proteins under
high salt condition and then elute the bound proteins by running a salt gradient from high to low
concentrations.
Electrophoresis
Electrophoresis is an analytic method to analyze the purity of proteins. It is can also be used to
estimate molecular weight of proteins.
In solution, charged molecules experiencing electrostatic potential will move towards the
opposite charged electrode. Electrophoresis is carried out in a porous support matrix such as
polyacrylamide or agarose which retard the movement of molecules according to their
dimensions.
Different proteins carry different charges that depend on pH of the solution. In order to
uniformly charge proteins and to disrupt protein folding, SDS or sodium dodecylsulfate is added
to the protein solution. Thus electrophoresis of proteins is usually called SDS-Polyacrylamide
Gel Electrophoresis (SDS-PAGE). The hydrophobic tail of dodecylsulfate interacts strongly
with polypeptide chains. Each dodecylsulfate contribute two negative charges. Thus all protein
samples undergone electrophoresis are negatively charged. Sulfhydryl-reducing agents such
as -mercaptoethanol is added in order to disrupt disulfide bond. The electrophoretic mobility of
proteins upon SDS-PAGE is inversely proportional to the logarithm of the protein’s molecular
weight.
SDS-PAGE Gel
Proteins on SDS-PAGE are stained by commassie blue.
Protein Sequencing Strategies (Chemical)
Before the advent of modern DNA technology, the sequencing of proteins was very laborious
and frequently inaccurate. In fact, many thought that it would be an insurmountable task.
In 1953, Frederick Sanger worked out the sequence of the amino acid residues that comprise
the polypeptides of the hormone insulin, for which he received the Nobel Prize. Note that
Frederick Sanger received another Nobel Prize later for developing a method for sequencing
DNA.
Sequencing by chemical methods
– If more than one polypeptide chain, separate.
– All disulfide bonds must be cleaved.
– Determine the amino acid composition of each of the chains.
– Determine N- and C-terminal residues by Edman degradation and C-terminal proteases
– Cleave each chain into smaller fragments by site-specific proteases and determine the
sequence of each chain by the above methods
– Reconstruct the sequence of the protein from the sequences of overlapping fragments
– Locate the positions of disulfide bonds.
重点：重点介绍蛋白质二级结构特征及等电点等主要性质,蛋白质分离纯化技术
难点：二级结构特点及蛋白质分离纯化技术的正确理解
教学过程设计（要求阐明对教学基本内容的展开及教学方法与手段的应用、讨论、作业布置）：
利用课件结合板书介绍蛋白质化学的基础知识，了解蛋白质化学的研究新进展，重点掌握蛋
白质二级结构特征及等电点等主要性质，在理解的基础上布置作业，让学生在作业中发现问题提
出问题，对于比较难理解的老师在课堂上再次强调。在教学过程中给学生介绍学习方法及鼓励学
生拓展知识。