x ray crystallography, Protein structure function relationship

1. [2019]
Structural Biology
[PROTEIN STRUCTURE BY X-RAY CRYSTALLOGRAPHY, PROTEIN STRUCTURE
AND FUNTION RELATIONSHIP]
SARDAR HUSSAIN, ASST. PROF., BIOTECHNOLOGY, GSC, CTA.

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Lecture notes SH/GSC/BT
1 Structural Biology
STRUCTURAL BIOLOGY
 Determination of protein structure
 x-ray diffraction
 Protein structure function relationship
DETERMINATION OF PROTEIN STRUCTURE AND X-RAY DIFFRACTION
BASICS OF XRD
Wilhelm Röntgen discovered X-rays in 1895, just as the studies of crystal symmetry were being
concluded. Physicists were initially uncertain of the nature of X-rays, but soon suspected (correctly)
that they were waves of electromagnetic radiation, in other words, another form of light.
Crystals are regular arrays of atoms, and X-rays can be considered waves of electromagnetic
radiation. Atoms scatter X-ray waves, primarily through the atoms' electrons. Just as an ocean wave
striking a lighthouse produces secondary circular waves emanating from the lighthouse, so an X-ray
striking an electron produces secondary spherical waves emanating from the electron. This
phenomenon is known as elastic scattering, and the electron (or lighthouse) is known as the scatterer.
A regular array of scatterers produces a regular array of spherical waves. Although these waves
cancel one another out in most directions through destructive interference, they add constructively in
a few specific directions, determined by Bragg's law:
The incoming beam (coming from upper left) causes each scatterer to re-radiate a small portion of its
intensity as a spherical wave. If scatterers are arranged symmetrically with a separation d, these
spherical waves will be in sync (add constructively) only in directions where their path-length
difference 2d sin θ equals an integer multiple of the wavelength λ. In that case, part of the incoming
beam is deflected by an angle 2θ, producing a reflection spot in the diffraction pattern
Here d is the spacing between diffracting planes, Theta is the incident angle, n is any integer, and λ is
the wavelength of the beam. These specific directions appear as spots on the diffraction
pattern called reflections. Thus, X-ray diffraction results from an electromagnetic wave (the X-ray)
impinging on a regular array of scatterers (the repeating arrangement of atoms within the crystal).

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Lecture notes SH/GSC/BT
2 Structural Biology
Analyzing Protein Structure and Function
Proteins perform most of the work of living cells. This versatile class of macromolecule is involved in
virtually every cellular process: proteins replicate and transcribe DNA, and produce, process, and
secrete other proteins. They control cell division, metabolism, and the flow of materials and
information into and out of the cell. Understanding how cells work requires understanding how
proteins function.
The question of what a protein does inside a living cell is not a simple one to answer. Imagine
isolating an uncharacterized protein and discovering that its structure and amino acid sequence
suggest that it acts as a protein kinase. Simply knowing that the protein can add a phosphate group
to serine residues, for example, does not reveal how it functions in a living organism. Additional
information is required to understand the context in which the biochemical activity is used. Where is
this kinase located in the cell and what are its protein targets? In which tissues is it active? Which
pathways does it influence? What role does it have in the growth or development of the organism?
In this section, we discuss the methods currently used to characterize protein structure and function.
We begin with an examination of the techniques used to determine the three-dimensional structure of
purified proteins. We then discuss methods that are used to predict how a protein functions, based
on its homology to other known proteins and its location inside the cell. Finally, because most
proteins act in concert with other proteins, we present techniques for detecting protein-protein
interactions. But these approaches only begin to define how a protein might work inside a cell. In the
last section of this chapter, we discuss how genetic approaches are used to dissect and analyze the
biological processes in which a given protein functions.
The Diffraction of X-rays by Protein Crystals Can Reveal a Protein's Exact Structure
Starting with the amino acid sequence of a protein, one can often predict which secondary structural
elements, such as membrane-spanning α helices, will be present in the protein. It is presently not
possible, however, to deduce reliably the three-dimensional folded structure of a protein from its
amino acid sequence unless its amino acid sequence is very similar to that of a protein whose three-
dimensional structure is already known. The main technique that has been used to discover the three-
dimensional structure of molecules, including proteins, at atomic resolution is x-ray crystallography.
X-rays, like light, are a form of electromagnetic radiation, but they have a much shorter wavelength,
typically around 0.1 nm (the diameter of a hydrogen atom). If a narrow parallel beam of x-rays is
directed at a sample of a pure protein, most of the x-rays pass straight through it. A small fraction,
however, is scattered by the atoms in the sample. If the sample is a well-ordered crystal, the scattered
waves reinforce one another at certain points and appear as diffraction spots when the x-rays are
recorded by a suitable detector (Figure 8-45).

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Lecture notes SH/GSC/BT
3 Structural Biology
(A) A narrow parallel beam of x-rays is directed at a well-ordered crystal
(B) Shown here is a protein crystal of ribulose bisphosphate carboxylase, an enzyme with a central
role in CO2 fixation during photosynthesis. Some of the beam is scattered by the atoms in the crystal.
The scattered waves reinforce one another at certain points and appear as a pattern
of diffraction spots
(C) This diffraction pattern, together with the acid sequence of the protein, can be used to produce
an atomic model
(D) The complete atomic model is hard to interpret, but this simplified version, derived from the x-
ray diffraction data, shows the protein's structural features clearly (α helices, green; β strands, red).
Note that the components pictured in A to D are not shown to scale.
(B, courtesy of C. Branden; C, courtesy of J. Hajdu and I. Anderson; D, adapted from original
provided by B. Furugren.)
The position and intensity of each spot in the x-ray diffraction pattern contain information about the
locations of the atoms in the crystal that gave rise to it. Deducing the three-dimensional structure of a
large molecule from the diffraction pattern of its crystal is a complex task and was not achieved for
a protein molecule until 1960. But in recent years x-ray diffraction analysis has become increasingly
automated, and now the slowest step is likely to be the generation of suitable protein crystals. This
requires large amounts of very pure protein and often involves years of trial and error, searching for
the proper crystallization conditions. There are still many proteins, especially membrane proteins
that have so far resisted all attempts to crystallize them.

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4 Structural Biology
Analysis of the resulting diffraction pattern produces a complex three-dimensional electron-density
map. Interpreting this map—translating its contours into a three-dimensional structure—is a
complicated procedure that requires knowledge of the amino acid sequence of the protein. Largely by
trial and error, the sequence and the electron-density map are correlated by computer to give the best
possible fit. The reliability of the final atomic model depends on the resolution of the original
crystallographic data: 0.5 nm resolution might produce a low-resolution map of the polypeptide
backbone, whereas a resolution of 0.15 nm allows all of the non-hydrogen atoms in the molecule to be
reliably positioned.
A complete atomic model is often too complex to appreciate directly, but simplified versions that
show a protein's essential structural features can be readily derived from it (see Panel 3-2, pp. 138–
139). The three-dimensional structures of about 10,000 different proteins have now
been determined by x-ray crystallography or by NMR spectroscopy (see below)—enough to begin to
see families of common structures emerging. These structures or protein folds often seem to be more
conserved in evolution than are the amino acid sequences that form them (see Figure 3-15).
X-ray crystallographic techniques can also be applied to the study of macromolecular complexes. In a
recent triumph, the method was used to solve the structure of the ribosome, a large
and complex cellular machine made of several RNAs and more than 50 proteins (see Figure 6-64).
The determination required the use of a synchrotron, a radiation source that generates x-rays with the
intensity needed to analyze the crystals of such large macromolecular complexes.
Protein Structure and Function (extract from L.Stryer)
Why do we need to understand protein structure and function relationship?

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Lecture notes SH/GSC/BT
5 Structural Biology
Proteins are the most versatile macromolecules in living systems and serve crucial functions in
essentially all biological processes. They function as catalysts, they transport and store other
molecules such as oxygen, they provide mechanical support and immune protection, they generate
movement, they transmit nerve impulses, and they control growth and differentiation. Indeed, much
of this text will focus on understanding what proteins do and how they perform these functions.
Several key properties enable proteins to participate in such a wide range of functions.
1. Proteins are linear polymers built of monomer units called amino acids.
The construction of a vast array of macromolecules from a limited number of monomer building
blocks is a recurring theme in biochemistry. Does protein function depend on the linear sequence of
amino acids? The function of a protein is directly dependent on its three-dimensional structure
(Figure 3.1). Remarkably, proteins spontaneously fold up into three-dimensional structures that are
determined by the sequence of amino acids in the protein polymer. Thus, proteins are the embodiment of
the transition from the one-dimensional world of sequences to the three-dimensional world of molecules capable
of diverse activities.
2. Proteins contain a wide range of functional groups.
These functional groups include alcohols, thiols, thioethers, carboxylic acids, carboxamides, and a
variety of basic groups. When combined in various sequences, this array of functional groups
accounts for the broad spectrum of protein function. For instance, the chemical reactivity associated
with these groups is essential to the function of enzymes, the proteins that catalyze specific chemical
reactions in biological systems
3. Proteins can interact with one another and with other biological macromolecules to form complex
assemblies.
The proteins within these assemblies can act synergistically to generate capabilities not afforded by
the individual component proteins these assemblies include macro-molecular machines that carry out
the accurate replication of DNA, the transmission of signals within cells, and many other essential
processes.
4. Some proteins are quite rigid, whereas others display limited flexibility.
Rigid units can function as structural elements in the cytoskeleton (the internal scaffolding within
cells) or in connective tissue. Parts of proteins with limited flexibility may act as hinges, springs, and
levers that are crucial to protein function, to the assembly of proteins with one another and with
other molecules into complex units, and to the transmission of information within and between cells .
A) Crystals of human insulin.
Insulin is a protein hormone, crucial for maintaining blood sugar at appropriate levels. (Below)
Chains of amino acids in a specific sequence (the primary structure) define a protein like insulin.

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Lecture notes SH/GSC/BT
6 Structural Biology
These chains fold into well-defined structures (the tertiary structure)—in this case a single insulin
molecule. Such structures assemble with other chains to form arrays such as the complex of six
insulin molecules shown at the far right (the quarternary structure). These arrays can often be
induced to form well-defined crystals (photo at left), which allows determination of these structures
in detail.
[(Left) Alfred Pasieka/PeterArnold.]
B) Structure Dictates Function.
A protein component of the DNA replication machinery surrounds a section of DNA double helix.
The structure of the protein allows large segments of DNA to be copied without the replication
machinery dissociating from the DNA.
C) A Complex Protein Assembly. An electron micrograph of insect flight tissue in cross section
shows a hexagonal array of two kinds of protein filaments. [Courtesy of Dr. Michael Reedy.]

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7 Structural Biology
D) Flexibility and Function.
Upon binding iron, the protein lactoferrin undergoes conformational changes that allow other
molecules to distinguish between the iron-free and the iron-bound forms.
Summary
 3.1 Proteins Are Built from a Repertoire of 20 Amino Acids
 3.2 Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains
 3.3 Secondary Structure: Polypeptide Chains Can Fold Into Regular Structures Such as the
Alpha Helix, the Beta Sheet, and Turns and Loops
 3.4 Tertiary Structure: Water-Soluble Proteins Fold Into Compact Structures with Nonpolar
Cores
 3.5 Quaternary Structure: Polypeptide Chains Can Assemble Into Multisubunit Structures
 3.6 The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure