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OCR for page 37
Report of the
Research Briefing Panel on
Protein Structure and Biological Function
OCR for page 38
Research Briefing Panel on
Protein Structure and Biological Function
Frederic Richards (Chairman), Yale
University, New Haven, Conn.
Robert Baldwin, Stanford University
Medical Center, Palo Alto, Calif.
Gerald R. Galluppi, Monsanto Company,
St. Louis, Mo.
Robert Griffin, Massachusetts Institute of
Technology, Cambridge, Mass.
Emil Thomas Kaiser, Rockefeller
University, New York, N.Y.
Brian Matthews, University of Oregon,
Eugene, Oreg.
l. Andrew McCammon, University of
Houston-University Park, Houston, Tex.
Alfred Recifield, Brandeis University,
Waltham, Mass.
Brian Reid, University of Washington,
Seattle, Wash.
Robert Saner, Massachusetts Institute of
Technology, Cambridge, Mass.
38
Alan Schechter, National Institutes of
Health, Bethesda, Md.
Paul Sigler, University of Chicago,
Chicago, Ill.
Peter van Hippel, University of Oregon,
Eugene, Oreg.
Don Wiley, Harvard University,
Cambridge, Mass.
Staff
Barbara FiIner, Director, Division of Health
Sciences Policy, Institute of Medicine
Naomi Hudson, Administrative Secretary
Allan R. Hoffman, Executive Director,
Committee on Science, Engineering,
and Public Policy
OCR for page 39
Report of the
Research Briefing Panel on
Protein Structure and Biological Function
INTRODUCTION
Proteins are involved in every biological
function. As enzymes, they catalyze the
chemical reactions of cells. As hormones and
growth factors, they regulate the develop-
ment of cells and coordinate the functions
of distant organs in the body. In various
filamentous forms, they control the shape
of cells and the dramatic alterations that oc-
cur during cell division. In muscle, proteins
change chemical energy into mechanical en-
ergy and cause movement. As components
of membranes, they control the traffic of
molecules anct information among the var-
ious cellular compartments. Hemoglobin, a
protein in blood, is specifically designed to
transport oxygen between organs; other
blood proteins, such as clotting factors and
circulating antibodies, act as defenses against
trauma and infection. In plants, a highly
organized collection of membrane proteins
is involved in the complex process of pho-
tosynthesis, without which there would be
no higher animal forms.
Proteins are polymer molecules com-
posed of amino acids that are connected by
links known as peptide bonds. An individ-
ual protein molecule may contain hundreds
39
or thousands of amino acids arranged in one,
or several, polypeptide chains. Each chain
folds into a particular three-dimensional
configuration that is essential for its highly
specific biological function. In this report,
we focus on the experimental and theoret-
ical investigation of the three-dimensional
structure of proteins, at the level of reso-
lution of individual atoms.
The study of protein structures, fre-
quently referred to as structural biology, is
in a period of great excitement brought about
by developments in several fields. Many
proteins of special interest are now available
in unprecedented amounts. The chemical
synthesis of polypeptide chains of increas-
ing size has steadily improved over the past
20 years, and recently a chain length of over
100 amino acids was achieved. In the past
5 years, the biological synthesis of proteins,
through cloning, has been reduced to stan-
dard practice, not only in the laboratory but
also on a commercial scale. These comple-
mentary procedures supply proteins of de-
fined sequence in substantial amounts. At
the same time, spectacular advances have
been made in x-ray diffraction and nuclear
magnetic resonance spectroscopy, two tech-
niques for determining structure. New pro
OCR for page 40
cedures and refined equipment have ex-
panded the range of application and the size
of the proteins that can be studied. Chem-
ical theory also has been developing at a
rapid rate, especially those branches related
to polymers. Furthermore, with the distri-
bution of faster, smaller, and less expensive
computer hardware, there has been con-
stant improvement in access to more ad-
vanced computing capability, a factor that
has played an important role in theoretical
and experimental studies. The coming to-
gether of these separate developments makes
structural biology ready for an explosive in-
crease in the determination and under-
standing of high-resolution structures of
proteins and protein complexes.
The new surge of structural information
will dramatically improve our understand-
ing of the processes of biological control and
will guide the design of proteins or other
products that will be developed either to
cause purposeful malfunctions (e.g., insec-
ticides) or to correct natural malfunctions
(e.g., to improve human health). Guided by
the three-dimensional structure, changes can
be introduced into the sequence of an en-
zyme that can alter the specificity of the cat-
alyzed reaction and/or its catalytic rate. In
addition, structural analysis may reveal spe-
cific differences in essential enzymes that
will enable geneticists to engineer protein
sequences, or drug designers to produce re-
agents, that will selectively counter harmful
bacteria or insects without harming the host.
RESEARCH GOALS
THE FOLDING PROBLEM
Inside a cell, amino acids are assembled
into peptide chains by a complex system
that translates the genetic message into spe-
cific amino acid sequences. Following syn-
thesis, the chains fold into compact protein
molecules. For many isolated polypeptides,
conditions can be provided in which this
folding step will occur spontaneously,
40
yielding a biologically active molecule iden-
tical to the native, ceD-derived protein. Thus,
all of the information required to produce
the final structure is contained in the amino
acid sequence. The prediction of the de-
tailed three-dimensional structure of a pro-
tein from a given sequence is known as the
folding problem. It is the most fundamental
problem at the chemistry-biology interface,
and its solution has the highest long-range
· -
priority.
The folding problem is not only a major
intellectual challenge but also an urgent and
immediate problem at the practical level in
biotechnology. Successful industrial pro-
duction of a biologically active protein fre-
quently depends on the ability to induce a
cloned polypeptide to fold correctly.
PROTEIN STABILITY
Protein stability Is a specific issue within
the folding problem. Thermodynamically,
protein structures are only marginally sta-
ble, and small changes can substantially in-
crease or decrease their effective stability.
Not only is prediction of stability a strin-
gent, but elusive, test of theoretical un-
derstanding, but also direct practical appli-
cations, through genetic engineering of pro-
teins, are immediately at hand. Resistance
to thermal destruction or to degradation by
enzymes secreted by microorganisms, for
example, are highly desirable properties for
pharmacological agents and enzymes in in-
dustrial use.
L~GAND BINDING
The specificity and strength with which
ligands, either small or large molecules, bind
to proteins is a central feature of biological
function. The result of this binding may be
simple sequestration for storage or removal,
a catalytic event if the protein is an enzyme,
the development and transmission of a sig-
nal if the protein is a receptor, or the switch-
ing off of a gene if it is a repressor. The
OCR for page 41
PROTEIN STRUCTURE AND BIOLOGICAL FUNCTION
ability to predict the structure either of a
protein ligand or an active site the part of
the protein where the ligand binds is cen-
tral to the rational design of new drugs or
new enzymes.
SIGNAL TRANSMISSION
Signal transmission from one part of a
molecule to another is essential for the reg-
ulation of complex enzymes and for the ac-
tivity of many receptor systems. Its
mechanism, however, is poorly under-
stood. Changes in the dynamic properties
of an entire protein molecule can be caused
by the binding of a ligand to a relatively
small active site; this phenomenon has clear
Implications for information transfer. In some
proteins, however, similar interactions pro-
duce only localized changes in structure or
dynamics. Full understanding will only come
through a detailed study of the relevant
structures and their properties.
RECENT ADVANCES IN KNOWLEDGE
About 300 protein structures are known,
and about 10 to 20 new structures are re-
ported each year. In many cases, knowledge
of these structures has brought us closer to
the goals outlined above understanding
folding, stability, ligand binding, signal
transmission, and catalytic activity. A few
examples will illustrate what has been
gleaned from studies of structure and the
potential applications of the knowledge.
Ang~otensin is a chemical in human blood
that is involved in regulation of blood pres-
sure. When it is modified by an enzyme
known as ang~otensin-converting enzyme
(ACE), it causes a rapict increase in blood
pressure. Control of high blood pressure
seemed possible if an inhibitor of ACE could
be found. At the time a drug development
program was started, ACE had not been iso-
lated in pure form from humans. But the
structure of an enzyme from the pancreas
of cows, which happens to catalyze a chem
41
ically similar reaction, was known at high
resolution. Based on the detailed structure
of the animal enzyme, especially the cata-
lytic binding site, it was possible to make a
mode! for the active site of human ACE.
With this model, the drug captopril, a strong
inhibitor of human ACE, was successfully
designed and synthesized. Subsequently,
protein chemistry was used to design ena-
lapril, a new drug in which some of the
unwanted side effects of captopri] have been
eliminated.
Influenza virus offers another example.
This virus causes recurring epidemics (and
the continuing need for development of new
vaccines) because its surface proteins vary
so much. Recently, the structure of hae-
magglutinin, a surface protein, was deter-
mined. Consequently, the regions that vary
with the strain of virus have been located.
Even more promising is the discovery of a
region that does not vary; it provides a pocket
in the protein and may be an excellent target
for drug development. The full high-reso-
lution structure was essential to the discov-
ery of this region. Recent structural studies
of the coIct and polio viruses and adenovi-
ruses have opened up similar exciting op-
portunities.
The recent determination of the structure
of a part of an enzyme called DNA poly-
merase I, in conjunction with related kinetic
studies, is beginning to lay a general foun-
dation for understanding how "processive"
enzyme reactions work. Such enzymes latch
onto a long molecule (the DNA thread in
the case of DNA polymerase) and then move
rapidly along it without letting go, much
like a train on a track. Such a mechanism is
entirely new in the field of enzymology. Not
only is this fascinating to biochemists study-
ing the replication of DNA and RNA of
central importance to life but the practical
importance is considerable. The processive
digestion of polysaccharides and other
macromolecules is of great importance to
food processing and pharmaceutical indus-
tries, for example.
OCR for page 42
Instrumentation clevelopments have been
essential to these and numerous other ex-
amples of major progress in structural bi-
ology. New or improved instrumentation has
led to tremendous savings of time and man-
power, as well as to unique routes for solv-
ing research problems. Accordingly, much
of the remainder of this report will focus on
research advances in instrumentation and
data analysis.
TECHNOLOGICAL ADVANCES
X-RAY DIFFRACTION
Since the early 1960s, x-ray crystallogra-
phy has continued to provide us with the
most detailed and comprehensive picture of
three-dimensional protein structure. When
x-rays are passed through a crystal, they are
diffracted in many directions, and the ge-
ometry and intensity of the many diffracted
beams are directly related to the structure
of the crystal. Improvements in x-ray sources,
in data collection equipment, and in the
power and availability of computers are ex-
pected to continue to enhance the power of
these structure studies.
Area Detectors
The diffraction pattern from a crystal can
be recorded all at once (with a photographic
fiIm) or one beam at a time (with an appro-
priate counter). Data on film must be read
optically and then must be converted to a
digital form to determine the intensity of
each beam. Area detectors are a major new
innovation in such data collection. They have
the advantage of direct counting while re-
taining the multiple recording capability of
film with a higher signal-to-noise ratio.
Analyses that took weeks or months have
been reduced to hours, with a considerable
· .
gain in accuracy.
This time-saving device has had far-
reaching effects on the kinds of experiments
that are being planned. For example, it will
42
now be possible to take full advantage of
the ability of molecular genetics to generate
many different mutational changes in a sin-
gle protein. The crystallographic examina-
lion of the different forms of a Oven protein
will be practical for a small group of inves-
tigators, or even a single individual. The
comparison of structures will be of inesti-
mable value in elucidating determinants of
structure and structure/function relation-
ships.
Synchrotron X-Ray Sources
Synchrotron x-ray sources are becoming
available at various national facilities, and
their accessibility provides unique oppor-
tunities. Because synchrotron radiation has
a continuously varying wavelength, it is
possible to collect data at two or more dif-
ferent wavelengths. Proper combination of
the data sets provides substantial help in
overcoming the major stumbling block in
solving an unknown structure.
The high intensity of the synchrotron ra-
diation also far exceeds that of any usual
laboratory source. It is possible to collect
enough data for structure definition in 10 to
100 milliseconds, and perhaps even faster
in the future. With such high data rates, full
structural studies of short-lived intermedi-
ates in enzyme reactions are possible in
principle. The determination of the struc-
ture of such intermediates at physiological
temperatures would lead to a dramatic im-
provement of our understanding of enzyme
catalysis. Currently, intermediate states can
only be studied when stabilized under un-
usual conditions, such as very low temper-
ature, so there is always uncertainty about
the relevance of any findings to the actual
catalytic process.
The full benefits of fast synchrotron data
collection are not yet being realized because
the diffracted intensities are recorded on
photographic film. More effective use of
synchrotron facilities for protein structure
investigations will require a hich-flux area de
O J
OCR for page 43
PROTEIN STRUCTURE AND BIOLOGICAL FUNCTION
sector capable of recording perhaps 108 events
per second. This is at least i,000 times faster
than the commercial instruments now avail-
able for laboratory use.
Computing and Graphics
X-ray studies require computers for data
analysis and interpretation. Mode! refine-
ment to get the "best" structure is particu-
larly computer-intensive. Computer-aided
molecular graphics plays an increasing role
in the solution of structure and in subse-
quent study of the structure. Color is rou-
tine and is now central to the effective use
of graphics as a laboratory tool. It is possible
to examine very complex structures and to
selectively "flag" special features or prop-
erties by color-coding the atoms. Spatial re-
lations that would be extremely difficult to
detect by computation become very obvious
to the human eye. The emphasis on graph-
ics is likely to continue, even with marked
improvements in automatic data analysis,
which is itself highly computer-intensive.
Neutron Diffraction
Neutron diffraction has a special role in
structure studies because of its unique abil-
ity to reveal the position of hydrogen atoms.
These atoms are frequently of central im-
portance in enzyme-catalyzed reactions, but
because hydrogen is so light, it is poorly
detectec! if at all in x-ray structures of
proteins. However, hydrogen is easily "seen"
by neutrons, although such experiments can
only be carried out at the national laboratory
reactors. Together with the hoped-for up-
grading of the high-flux reactors, the de-
velopment and capabilities of the new pulse
neutron source at Los Alamos win be watched
with great interest.
Crystallization
Protein crystals are unusual examples of
the solid state in that they contain a large
43
amount of liquid water. The structure of those
proteins for which comparisons have been
made is essentially identical in a crystal and
in aqueous solution.
Production of the highly ordered crystals
required for x-ray diffraction studies re-
mains an art rather than a science. None-
theless, the number of crystallized proteins
is increasing rapidly. Of special note is the
recent successful crystallization of integral
membrane proteins such as the photosyn-
thetic reaction center and bacterial rhodop-
s~n.
NUCLEAR MAGNETIC RESONANCE
With a nuclear magnetic resonance (NMR)
spectrum, researchers measure the absorp-
tion of radio-frequency energy by the nuclei
of molecules placed in a magnetic field. The
frequency at which an atomic nucleus ab-
sorbs radiation is very sensitive to the chem-
ical environment provided by the structure
of the molecule. A basic problem, however,
has been the identification of which peak in
the spectrum belongs to which atomic nu-
cleus in the structure. New data collection
techniques have been developed to extract
information about the distances between
neighboring atoms, and these techniques
have revolutionized the study of proteins
up to approximately 12,000 in molecular
weight. From these identified spectra, sci-
entists can derive characteristic patterns of
substructures in the protein. More detailed
analysis with computer-intensive distance
geometry algorithms can provide the full
three-dimensional structure in favorable
cases.
The recent progress in research on small
proteins has been directed toward the de-
termination of average structures in solution
for comparison with models from x-ray dif-
fraction. This work has set the stage for the
next fascinating phase of NMR, the study
of changes in structure that are induced, for
example, by the binding of ligands. While
much of this work will be done in partner
OCR for page 44
ship with investigators using x-ray diffrac-
tion, many problems are only accessible
through NMR procedures notably those
cases in which crystalline materials cannot
be obtained. Unique opportunities exist to
learn about partially or totally disordered
molecules that are important both in equi-
librium populations and as reaction inter-
mediates.
Defining procedures for the precise ma-
nipulation of nuclear spin in a molecule-
spin engineering will continue to play an
important role in the development of op-
erating procedures for NMR spectrometers,
especially for macromolecules with their
complex spin systems. An appropriate se-
quence of radio-frequency pulses can dras-
tically simplify a complex spectrum, reveal
relations between spatially distant atoms,
and greatly assist in the essential step of
assigning peak signals to portions of the
protein structure. Further developments re-
quire that young investigators with a back-
ground in quantum physics be attracted to
this particular area of structural biology.
Several other techniques designed for
structures larger than those with molecular
weights of 15,000 to 20,000 have also been
developed. Solid-state NMR has no inher-
ent size limit, and there are very interesting
applications for membrane proteins or fi-
brous materials, such as collagen, which are
intrinsically insoluble. Another approach is
the direct study of small substrates or in-
hibitors interacting with active sites of large
enzymes. A number of new developments
are being intensively pursued in this area,
such as the use of labeled, tightly bound
substrates. A third approach is to simplify
the spectra by preparing samples with sta-
ble isotopes inserted in a limited number of
known positions. By a combination of
chemical and biological procedures, amino
acids are prepared with the isotopes 2H, 13C,
or i5N in appropriate positions. These are
incorporated into proteins at known loca-
tions. The syntheses are often difficult, but
the rewards are great because the spectra of
44
the isotopically substituted proteins can be
very simple and easy to interpret. More-
over, this technique can produce very large
signals in comparison with the low back-
ground absorption. Even low concentra-
tions of relatively unstable intermediates,
such as are likely to be important in the
protein folding problem, may be detectable
in these enriched samples. And as ~ further
benefit, data collection times are markedly
shortened.
SYNTHESIS OF PROTEINS
Sensitivity and sample size continue to
limit both x-ray crystallography and NMR,
which require amounts of material in the 10-
to lOO-mg range. An adequate quantity of
highly purified proteins of specified se-
quence, and, where required, with specific
isotopic substitutions, is essential to bio-
physical study of structure and function.
Within different but overlapping size ranges,
quantities of proteins can be produced to-
day either by chemical or biological proce-
dures.
Chemical Synthesis
Solid-phase chemical synthesis has been
effectively automated, and peptides 30 to 40
amino acids long are readily produced in
good yield. Substantially longer peptides also
have been synthesized, and continuing im-
provement can be expected. The next step
toward the synthesis of longer chains is the
condensation of preformed fragments. Con-
densation is possible by enzymatic as well
as chemical methods, but general proce-
dures are not as well worked out and de-
serve considerably more study.
Chemical synthesis allows the insertion
of an isotopically labeled amino acid, an
amino acid derivative, or even a nonnatural
amino acid in any single position in the chain.
Multiple-site "mutations" at any selected
group of sites thus become easy to produce.
In the synthesis of drugs that mimic pep
OCR for page 45
PROTEIN STRUCTURE AND BIOLOGICAL FUNCTION
tides, even the peptide bond may be cir-
cumvented in specified locations, leading to
resistance to degradation.
Limitations in chemical synthesis at this
time derive from the problem of optical pu-
rity, the yield of the correct sequence, and
the roughly linear relationship between the
amount of the product and the cost of pro-
ducing it. However, these limitations, as wed
as the limitation on chain length, are over-
come in some biotechnology processes.
BioZogicaZ Synthesis
The procedures that are used to produce
proteins in high yield by cloning in bacteria
are well developed. The companion proce-
dures for producing single amino acid
changes by site-directed mutagenesis are
simple, fast, and reliable. In many cases, the
fusion of a special "signal sequence" to the
protein will result in its secretion, which as-
sists in proper folding. Nonetheless, clon-
ing is not always successful. Degradation of
the product can severely reduce the yield;
inside the bacterial cell, the chain may not
fold to yield the desired, biologically active
molecule; and modification of certain amino
acids after polymerization of the protein as
required for various eukaryotic proteins
("post-translational" modification) does not
occur in bacteria.
Cloning in eukaryotic systems, particu-
larly human cell culture, is not yet as well
developed and is very expensive for pro-
ducing proteins in commercial quantities.
Improved expression vectors to overpro-
duce the desired protein, pro/ease-deficient
strains, rapid lysis methods, and better bio-
chemical separation procedures are all re-
quired. Two important research opportunities
are development of better methods for muZtipZe
site-directed changes at positions widely sepa-
rated in the sequence, and the deveZopment of
high expression systems for eukaryotic proteins.
it is equally important that these new pro-
tein products be characterized rapidly. Pre-
liminary structure evaluations by standard
45
biophysical procedures, particularly the var-
ious forms of optical spectroscopy, enable
screening of protein products and selection
of the most interesting for detailed study by
x-ray and NMR techniques.
THEORY
Theoretical studies of proteins are only
beginning to have a real impact in relation
to biological function. The field has been
stimulated by advances in computer tech-
nology, theoretical chemistry, and experi-
mental biochemistry. It is clear that theoretical
studies will play a major role in the design
of new proteins and of molecules with which
proteins interact.
The most highly developed theoretical
methods involve molecular dynamics sim-
ulations, in which computers are used to
simulate atomic motions in a protein and its
surroundings. When combined with a new
approach giving thermodynamic parame-
ters for reactions, dynamic simulations can
be used to make predictions concerning rec-
ognition and binding among proteins and
other molecules. This method has recently
been used successfully to calculate the af-
finity in an enzyme-inhibitor interaction; it
has promise for studies of protein folding,
stability, covalent reactivity, and noncova-
lent association. Practical applications in-
clude the design of drugs, enzymes,
antibodies, and other molecules.
The rates and mechanisms of enzyme-cat-
alyzed reactions and ligand binding are po-
tentially accessible through molecular
dynamics. A modification known as Brown-
ian dynamics is useful for extending calcu-
lations into the time range of somewhat
slower biological processes. A number of
other theoretical approaches, clearly on the
horizon, may be useful for predicting the
structures of short peptides in solution.
Continuing attention should be directed
to improving the basic mathematical func-
tions and input parameters that are needed
both in molecular dynamics and energy
OCR for page 46
minimization procedures; to improving the
treatment of electrostatic interactions; and
to the detailed treatment of the water-pro-
tein interface where biological activity is ex-
pressed. More ad hoc approaches in applying
other aspects of basic chemical theory may
also be useful in attacking the protein fold-
ing problem in which the proper application
of first principles is still elusive.
BOTTLENECKS AND
RECOMMENDATIONS
Progress in solving the scientific problems
of structural biology will require both per-
sonne} qualified in this multidisciplinary area
and sophisticated equipment in individual
laboratories and national centers. For struc-
tural biology to achieve its full potential in
contributing to fundamental science, med-
icine, agriculture, and the chemical indus-
tries, a number of policy concerns should
be addressed.
I. Basic Research Support Of absolutely
central concern in this area, as in others, is
maintenance of support for basic research pro-
grams at the level of the individual investi-
gator and small consortia.
2. Professional Personnel Of comparable
concern is the continuing supply and training
of professional personnel. Scientific opportun-
ities will not be realizect, and the equipment
initiatives suggested below will have little
effect, if trained personnel are not available.
In the recent past, there has been a relatively
small number of individuals entering bio-
physics, biophysical chemistry, and the
general field of structural biology. The ma-
jor attraction during that period was clearly
molecular genetics. During the past year or
two, there has been an increasing number
of entering graduate students interested in
quantitative structural studies.
This student interest has coincided with
a dramatic upsurge in activity in the indus-
trial sector. A substantial number of bio-
technology firms have set up structural units,
46
including x-ray crystallography, high-reso-
lution NMR, and theoretical modeling. Re-
searchers experienced in one or more aspects
of structural biology and general protein
chemistry are in high demand at this time,
and corporations have attracted much of the
presently available talent.
Currently available predoctoral training
programs may be adequate to provide the
necessary graduate student input to this field
provided these predoctoral training programs are
maintained at a level at least equal to their present
levels. A particularly effective source of highly
qualified personnel is provided by the Med-
ical Scientist Training Program. If there are
further cuts in any of the programs, the
structure area, which is poised scientifically
for substantial progress, will be nipped in
the bud, and will suffer proportionally more
than the well-populated areas of molecular
and cellular biology.
The most serious concern for personnel
is postdoctoral training. The new genera-
tion of structural biologists must be familiar
with molecular biology as well as biophys-
ics, and it would be highly desirable that
molecular biologists with an interest in
structure learn at least the rudiments of the
biophysical methods. Similarly, physicists
and instrumentation engineers whose ex-
pertise could be shifted rapidly to structural
biology must learn some molecular biology.
This interdisciplinary training is difficult to
accomplish properly in the time period of a
normal doctoral program; postdoctoral
training thus becomes even more essential
than usual under these circumstances. We
urge that the postdoctoral fellowship programs
be maintained and, if possible, expanded to cover
the present and anticipated needs in structural
biology.
3. Supply and Development of Major
Instruments The entire field of structural
biology is heavily dependent on major
equipment items and on unique facilities
available at certain national centers. Contin-
uing progress on the biological problems in
this field will be closely correlated with the
OCR for page 47
PROTEIN STRUCTURE AND BIOLOGICAL FUNCTION
improvement and availability of advanced
instrumentation.
X-Ray Diffraction Currently available area
detectors are having an enormous impact
on the efficiency of data collection and on
the types of research programs that it is re-
alistic to plan. Agencies should! be prepared to
fund acquisition of area detectors, and anciliar~u
equipment for efficient utilization, widely
throughout the structural bioZo~u community over
the next few years.
cam ~
The effective use of the synchrotron x-ray sources
at the national laboratories will depend on the
development of high-flux area detectors capable
of recording at least 108 events per second. Ef-
forts are uncler way for other scientific fields;
the needs of structural biology should be
consiclered as part of this general develop-
ment effort.
Nuclear Magnetic Resonance The distri-
bution of 500-MHz instruments, or their fu-
ture replacements, will continue to present
a policy problem. Laboratories devotee! to
the development of NMR techniques, or to
major long-term protein-structure projects,
win need fully dedicated instruments of their
own. Shared facilities should still be dedi-
cated to the study of macromolecules and
should not be expected to provide small
molecule spectra as an acIditional service
component.
Improvements in resolution and sensitiv-
ity will depend on the development of
stronger magnets. Sensitivity and resolu-
tion both become increasingly important as
the size of the protein increases, and both
are improved as the magnetic field strength
of the spectrometer is increased. We suggest
that a major effort be launched to interest and
encourage instrument companies to produce
spectrometers with a 17.5 T (750-MHz) magnet.
This appears to be feasible with currently
available superconducting wire technology,
although some engineering difficulties re-
main to be solved. A magnet at 20-25 T
(~l,000 MHz) is not out of the question,
although an intensive investigation in the
materials science area may be involved. Suc
47
cess, however, would have a dramatic im-
pact on biophysical NMR studies.
Availability of precursor chemicals la-
beled with stable isotopes is another NMR
concern. The very high price of labeled ma-
terial is a serious general problem for sci-
entists. The Stable Isotope Facility at Los
Alamos is bound by regulation to stop any
activity that is taken up in the private sector.
In contrast to many other examples of sci-
entific resource supply, this particular reg-
ulation has worked to the clisadvantage of
the research community. There is not a suf-
ficiently large market for labeled com-
pounds to reduce the price through volume
and competition. Even the development of
clinical applications would produce a mar-
ket for only a relatively small number of
compounds and would not cover the broad
range neecled for the research proposed
above. It is essential that a mechanism be found
for providing amino acids and nucleotides with
a variety of different stable isotope labeling pat-
terns for the research community.
Computers The instrumentation needs for
theoretical and experimental studies of pro-
teins include increased and predictable access to
supercomputers; improved communications
between these machines anct remote users
through networking; and additional access
to high-quality graphics devices, scientific
workstations, and special-purpose com-
puters.
X-ray crystallography, NMR, and theo-
retical studies all have very heavy com-
puting requirements. The larger the
molecules and the faster the data collec-
tion, the larger will be the computational
requirements in the immediate future. Even
for structures of modest size, the current
iterative x-ray refinement procedures cre-
ate major inconvenience to other users of
a VAX. (Refinement cycles can occupy tens
of hours per cycle.) Protein data process-
ing even from current NMR spectrometers
requires levels of computation not nor-
mally available in the laboratories of in-
dividual investigators. Many theoretical
OCR for page 48
problems are not at all practical on inter-
mediate-leve} machines.
We estimate that the needs of the current
scientific community in structural biology
nationwide may already exceed the com-
puting power represented by two ad-
vanced-leve! Cray machines. The present
National Science Foundation Supercompu-
ter Center Program is useful in providing
access to these machines. This initiative,
however, covers all areas of science and may
wed become saturated. The necessary phased
expansion of this program should include
the present and anticipated requirements of
the structural biologists. A library of pro-
grams specifically written to take advantage
of the architecture of these machines also
will be essential for their effective use.
The efficient use of the supercomputers
will depend on the ease and convenience of
access. The latter will depend on the speed
and effectiveness of the networking that is
available to make this possible. Networks
suitable for connection of the lower-level
48
computers in the various structure labora-
tories also will become increasingly useful.
Experience over the next year or two with
the currently developing general purpose
networks will show whether or not they are
adequate for this purpose.
A large volume of protein sequence and
structure data can be expected in the near
future as a result of the many new metho-
dologies. Therefore, it is time to plan for the
accession and use of the ciata in a comput-
erized central data bank. Computer searches
of genetic structure data banks have pro-
vided significant new insights into biologi-
cal phenomena, and a similar outcome can
be expected from the protein structure data.
Finally, there will be increased need for
dedicated microcomputers and various lev-
els of graphics workstations in individual
laboratories. Although these are not ex-
pected to be particularly expensive, the need
for their wide distribution is clearly visible
now and should have a prominent place in
the planning for future equipment support.
Representative terms from entire chapter:
biological function
