Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Metalloprotein Techniques

395 Pages·1993·4.4 MB·English

by  Christopher Jones

60 Neuhaus and Evans
studies of protein-ligand complexes, for example, one can focus on
the ligand (see Chapter 7). Thus, one can study ligands by hetero-
nuclear NMR methods if they are labeled or, in the case of metal ions,
if they can be substituted by ions, such as ’ 13Cd2+t,h at can be studied
directly by NMR. For example, in studies of metallothionein, it was
possible to determine which residues coordinate the metal ion by detect-
ing coupling of cysteinyl CPH protons to li3Cd2+ (47). Alternatively,
it may be possible to study the conformation of the bound ligand when
it is in equilibrium with the free form (which may be in excess, so that
its spectrum is readily observed) by the detection of transferred NOES
(48). It may also be possible to use labeled ligands to obtain structural
information about the residues of the protein itself, to which they are
bound. NOES can also be detected between nuclei of the ligand and of
the protein, potentially providing a very powerful specific probe of the
binding site; however, the success of such experiments has so far
proven to be limited m practice, principally because they do not over-
come the problem of assigning the protein resonances concerned.
NMR also has considerable potential as a technique for studying
nonnative states of proteins. This is of considerable importance in
protein folding studies, and even though the information available
may be rather limited, it is, in most cases, virtually the only residue-
specific structural information obtainable and therefore very valuable.
The problem with partially unfolded states is that they tend to give
very poorly resolved spectra, so that direct assignment and structural
interpretation are very difficult. However, it may be possible to use the
well resolved native state spectrum to obtain mformation indirectly
about the nonnative one (8,49). For example, chemical shifts of indi-
vidual protons in the partially folded state may be determined by mag-
netization transfer from the corresponding native stater esonancesw here
the two forms are interconverting. Hydrogen bonded structure in the
partially folded form may be detectable by means of the protection it
offers against exchange of NHs for deuterons when the protein is
dissolved in D20. The pattern of labeling in the partially folded form
can be determined by allowing the protein to fold and determining the
extent of proton occupancy at individual sites m the well resolved
spectrum of the native form. This idea has now been extended to the
characterization of transient structural intermediates on refolding
pathways (50,51).
Proteins in Solution 61
7. Practical Considerations
The feasibility of undertaking a detailed structural study of a protein
by NMR depends, in part, on the intrinsic properties of the protein, as
discussed in Section 2. A full 3D structure determination generally
requires a level of assignment and analysis that is only currently attain-
able forrelatively small proteins. Thus, the NMR spectroscopist’s first
question about a protein is always said to be “how big is it?” As we
pointed out before, there are no hard and fast rules; as a guideline, we
would suggest that if the mol wt is cl0 kDa, it is a possibility well
worth considering, although only preliminary studies to gage the qual-
ity of the spectrum can really tell. For proteins between 10 and 15 kDa,
it may still be possible, but the undertaking becomes increasingly
onerous. A few spectra of proteins of this size have been assigned
using only homonuclear ‘HNMR, although in these cases, other tricks
were generally used to obtain “edited” spectra in order to resolve
problems of resonance overcrowding- for example, differential sol-
vent exchange rates of amide protons in the case of lysozyme (13) and
the variable oxidation state of the prosthetic group in the case of flavo-
doxin (I 7). In the caseo f proteins much larger than approx 15 kDa, 13C
or 15Nl abeling to permit heteronuclear studies will undoubtedly become
necessary to permit much progress with assignment to be made. The
alternative with these and still larger proteins is to settle for seeking
more limited structural information, as discussed in Section 6.
The other major limitation of NMR is its Insensitivity. Obtaining a
1- mit4 solution, the minimum desirable for ‘H NMR, requires 5 mg of
protein to be dissolved in a OS-mL sample, in the case of a protein of
10 kDa. The overall requirement, both in terms of the amount of pro-
tein needing to be purified and its solubility, could therefore in some
casesb e excessive. It is also important to note that the protein must not
only be soluble at this concentration, but it must also not aggregate
appreciably; otherwise, the effective mol wt will of course be greatly
increased, and the spectrum will correspondingly tend to be poorer. It
may be necessary to experiment with a variety of solution conditions
m order to optimize the spectral quality obtainable.
In preparing a sample for NMR studies, several factors unique to
this technique need to be borne in mind. In particular, the solvent
conditions may have to be adjusted. NMR studies require that the
62 Neuhaus and Evans
solvent water be at least 10% deuteratedt o permit the field frequency lock
to function; for some studies, it may be desirablet o work in virtually 100%
D,O. Thus, some form of buffer exchange is generally necessary.S ince
NMR samples are typically more concentratedt han those used for other
studies, this is usually associatedw ith a concentration step. The simplest
method is to freeze-dry the protein, in the absenceo f added buffer salts,
and then redissolve the product in a buffer appropriate to NMR work.
Some proteins cannot be freeze-dried, however, and m that case, it may
be necessary to use some form of concentrator that employs a semi-
permeable membrane to effect buffer exchange and concentration.
A particular problem with protein samples is the presence of small
molecule signals that can interfere with the spectrum. The most obvi-
ous of these is water. Since it is in general necessary to work in H,O
rather than D20 solution, in order to observe all of the exchangeable
NH signals, considerable effort has been put mto developing methods
for suppressing the water peak in protein spectra. This may be achieved
either by selective saturation of the water or selective excitation of
the remainder of the spectrum (52). Whatever technique is applied, the
key to successi s excellent field homogeneity, since line-shaped istortions
can lead to poor suppression of parts of the peak, resulting m serious
baseline distortions in the protein spectra obtained. It must be remem-
bered that any solute molecule contaimng protons will also give rise to
signals in the ‘H spectrum and, therefore, that it is desirable to remove
such species as far as possible. This can usually be achieved by dialy-
sis or gel filtration, but it can present more of a problem in relation to
the buffer requirements of the particular protein. The simplest solution
is to use an inorganic buffer, such as phosphate, whose only protons
are in fast exchange with the water and can readily be preexchanged
for deuterons if need be simply by freeze-drying from D20 solution;
alternatively, several common buffer salts are available asp erdeuterated
derivatives-simple compounds, such as (d,)-acetic acid are indeed
quite cheap to obtain. If it is necessary to use a protonated buffer, its
concentration should be kept to an absolute minimum.
It is desirable, as with all experiments, to minimize contaminants of
any kind, but certain types present particular hazards to NMR samples,
and it may be necessary to take special steps to avoid them. Paramag-
netic impurities can cause broadening and shifting of resonances in
protein spectra, and need to be excluded if they are found to be present
Proteins in Solution 63
in a protein sample. To remove trace metal ions, it may be sufficient
to add a low concentration of EDTA or EGTA to the solution, but it is
probably preferable to remove them altogether by dialysis against one
of these agents or passing the protein through a column of a metal ion
sequestering resin, Of course, the problem may need more careful
consideration in the case of a metalloprotein! It should be noted that
molecular oxygen is itself aparamagnetic impurity, and some workers
remove dissolved oxygen from NMR samples by freeze-thaw meth-
ods. However, the effects are rather marginal in practice, and since the
process is time-consuming and may have deleterious effects on some
proteins, it is not very common today.
Optimum linewidths in NMR spectra depend on a number of fac-
tors. One is the condition of the sample, which should be free of extrane-
ous matter. This can usually be achieved by centrifugation prior to
placing the sample in the NMR tube. Most important of all is the
homogeneity of the magnetic field, which must be optimized by “shim-
ming.” This may be a very tedious process (although increasingly it is
possible to get the instrument to do it, at least in part, automatically),
but is absolutely necessary. Some workers fmd that it is best to shim
using a sample of a small molecule first, where resolution of very fine
couplmgs provides a very stringent test of homogeneity-it should
then only be necessary to make small final adjustments on introducing
the protein sample. Another factor that is a key to the success of 2D
experiments is stability; this is to some extent dependento n the quality and
situation of the instrument, but care should also be taken by the experi-
menter to ensure, for example, that the probe temperature is fully equili-
brated before commencing acquisition. Running the experiment without
spinning the sample also improves stability substantially, without degrad-
mg the resolution noticeably, provided the shimming is adequate.
The discussion presented here can serve only to provide a brief
introduction to the study of protein structure by NMR. Today NMR is
becoming an increasingly accessible technique, no longer solely the
preserve of specialist spectroscopists. Nonetheless, it should be clear
that an NMR study, particularly at the level of detailed assignment and
structural analysis, is still a major undertaking requiring a considerable
input of time, far beyond that requiredj ust to acquire the spectra, and that
a degree of acquired expertise is necessary to obtain useful spectra and
interpret them correctly. Protein NMR is still far from being a routme
64 Neuhaus and Evans
technique, but the opportunities are constantly increasing to take advan-
tages of the unique structural information that it can generate.
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CHAPTER3
Peptide Structure
Determination by NMR
Michael I? Williamson
1. Introduction
The difference between peptides and proteins (the subject of
Chapter 2) is that peptides are molecules too small to have a “globu-
lar” structure. This means that the spectral assignment process is often
much simpler for peptides than it is for proteins, because there are
fewer signals present m peptide spectra; on the other hand, peptides
seldom adopt a single, well defined structure in solution, which makes
the interpretation of structural data more contentious for peptides than
it is for proteins. The emphasis in this chapter is therefore different
from that in Chapter 2. The acquisition of structurally relevant data is
straightforward, given a familiarity with modern two-dimensional (2D)
NMR techniques and is given less emphasis here, but the analysis of
the data is seen as the key to obtaining a meaningful answer, and is the
area where experience and expertise are most necessary.
The difficulty in dealing with flexible structures by NMR derives
from the fact that intramolecular motion (i.e., rotation about single
bonds) causes most NMR parameters, such as NOE, coupling con-
stant, and chemical shift, to be averaged, rather than giving a superpo-
sition of values, as IS seen in many other branches of spectroscopy.
This has a number of consequences. First, it is not at all obvious from
inspection of a spectrum whether one conformation or many confor-
From Methods m Molecular Biology, Vol 17 Spectroscop/c Methods and Analyses
NMR, Mass Spectrometry, and Metalloprotem Techmques Edited by C Jones, B Mulloy,
and A H Thomas CopyrIght 01993 Humana Press Inc , Totowa, NJ
69