concept/ in biochemi/ try
Edited by: WILLIAM M. SCOVELL Bowling Green State Universily Bowling Green, Ohio 43403
Nuclear Magnetic Resonance Spectroscopy in Biochemistry Steve Cheatham College of Pharmacy, University of Toledo, 2801 W. Bancroft, Toledo, OH 43606 Traditionally, structures of nucleic acids and proteins have been prohed by X-ray techniques ( I ) . X-ray fiber diffraction and X-ray crystal structures have been invaluable to our understanding of the structure and function of macromolecules. The basis for the genetic code inherent in the structure of DNA was determined bv model buildine based upon X-ray fiher diffraction patterns. The different confurmational forms of DYA (A. . . B,. and Z DNA) and manv orotein structures have been elucidated by x-ray crystallographic analysis. One of the questions often raised about structures derived from X-ray crystal analysis is how the structure in the crystal relates to the solution structure of the molecule. Crystal structures provide static representations of molecules, while molecules in solution are in constant motion. Furthermore, the crystal structure may be distorted by crystal packing forces. Because of the problems outlined above, techniques that can analvze molecular structure in solution are valuable aids to understanding how molecular structure relates to function. The increased availabilitv of inexuensive minicomouters coupled with the advent o~comme~cially available h&hfield superconducting magnets has added an important series of experimental nuclear magnetic resonance (NMR) techniques that can vield precise information about the structural and dynamic characteristics of macromolecules. Currently, the magnetic field strengths of commercial spectrometers with superconducting magnets range from 4.7 to 14.1 Tesla providing proton resonance frequencies from 200 to 600 MHz. Higher field strength creates greater sensitivity, and most experiments on macromolecules are performed on 400400-MHz instruments. This review will discuss the nature of the NMR experiment, the techniques used, the types of structural and dynamic information one obtains, and how one can view and refine structures using computer graphics techniques in combination with NMR data. The NMR Experlmenl Nuclear magnetic resonance spectroscopy, like many otber forms of spectroscopy, depends upon the absorption of energy by the nuclei of the atoms in the molecules being studied. The nuclei of many atomic isotopes absorb electromagnetic radiation in the radio-frequency region when placed in a magnetic field. Examples of NMR active nuclei include hydrogens ('H), deuterium PH), carbon-13 (I"),
phosphorus-31 (3lP) and nitrogen-14 (14N) and -15 (15N). This discussion will focus on the use of proton NMR ('H NMR) techniques to study solution macromolecular structure where the term proton refers to the hydrogen isotope of mass unit one. High-field NMR experiments are performed by a technique known as Fourier transform NMR (FT-NMR). The signal from a proton is detected in FT-NMR as a decaying sinusoidal wave known as the free induction decay (FID). The signal is a time-dependent function and the spectrum is referred to as a time domain spectrum. NMR data are normally displayed as frequency domain spectra. Fourier transformationof the raw data performs the conversion from time to frequency domain. A simple NMR spectrum consists of a plot of signal frequency vs. peak intensity. The relative intensity or area of a neak in the NMR snectrum is oro~ortional to the number of . . nuclei in thegroupahsorbing at thnt frequency. This means, for example, that the three protonsoiaCH, group will give a signal that is three times the intensity uf'asingle proton. The position of a ~ e a in k the NMR spectrum is dependent on the magnetic eniironment of the ploton. The magnetic field of an NMR instrument is static. Therefore the protons in a 500-MHz spectrometer absorb electromagnetic radiation at approximately 500 MHz. Fortunately, all protons do not absorb energy a t exactly the spectrometer frequency. The presence of the electron clouds of the protons in a molecule induce secondary magnetic fields that change the actual induced field at the nuclei. Thus the frequencies of protons in a molecule are shifted relative to the transmitter freauency by a few parts per million (ppm). The frequency a t i h i c h nuclei absorb radiation is termed the resonance freauencv. . . Each type of proton in a molecule usually exhibits a unique resonance frequency. The resonance frequencies of protons are reported relative to an internal or external standard in parts per million, and the frequency of a given proton relative to the standard is known as the chemical shift of that proton (denoted by the Greek character 6). The most commonly used internal standard for nonaqueous samples is tetramethylsilane (TMS). TMS is not water soluble, and aqueous samples of biomolecules are usually referenced to the methyl protons of either 2,2-dimethyl-2-silapentane-5sulfonate (DSS) or 3-trimethylysilylpropionate-2,2,3,3,d4 (TSP). The protons of the standard are set by convention to be 0 ppm. A typical proton NMR spectrum of tryptophan (Naf Volume 66 Number 2
February 1989
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Karplus Relationship
Figure 1. Proton NMR specbum of tryptophan In D20. The structure and numbering system of tryptophan are indicated in lhe Inset. The assignments of the proton resonances of tryptophan are indicated abave lhe peaks. Signals from the protons on nitrogen are not present because afexchange wiih D20.
form) obtained in DzO and referenced to external DSS is shown in Figure 1. The signals of tryptophan fall in two regions. The upfield signals between 3.6 and 2.8 ppm are from the aliphatic a and 0 protons. The downfield signals between 7.0 and 7.8 ppm are from the aromatic protons (Fig. 1).The laree sienal in the center a t 4.8 oom is due to residual H~O or H ~ i;the D sample. The protonion nitrogens undereo raoid exchanee with the solvent. DvO. and are renlaced bv z e u t k u m , whch does not ahso& Fadiation a t the same frequency and is not detectable in proton NMR experiments. Each of the nonexchangeable protons of tryptophan is represented by a signal in the NMR spectrum. Each proton signal (except Hp) consists of a series of lines. The signal for each of the 0protons, for example, consists of four lines. The multiplicity of lines observed for each proton is due to proton-proton coupling. Coupling arises as a result of interactions between the electrons of one proton and the electrons of neighboring protons. Because it is associated with interactions between the electrons surroundine nuclei. coupling occurs through bonds, and coupling between protons is usuallvnot detectable if the orotons are senarated bv four or coupling between nuclei is more bonds. The degree termed the coupling constant and for protons is typically between the range of 0-15 Hz. The coupling constant is just that, a constant, and does not change with increasing or decreasing magnetic field strength. Thus two protons with a coupling constant of 8 Hz have the same coupling in both a 200-MHz spectrometer and a 500-MHz spectrometer. Analysis of the proton-proton coupling networks in macromolecules is crucial to any attempt to determine the threedimensional structure of the molecule. The extent to which two nuclei couole is deoendent on several factors. The most important consideration for this discussion is the angular deoendence on the deeree of couoline. The aneular d e ~ e n dence of the couplingconstant can ce expressed quantitatively by the Karplus equation, which can he visualized graphically in Figure 2 (2). Currently, there are many different forms of the original Karolus equation, each one containing parameters that providk better fit between calculated and experimental data in a given system. In particular, a recent variation introduced b; Altona and co-workers permits analysis of the ribose and deoxyribose rings in RNA and DNA (3).The equation extends the original Karplus relationship by specifically including a correction term to account for the influence of electronegativity on the threebond coupling constant PJHH)within sugars.
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of
The parameters PI-P6 are empirically determined based 112
Journal of Chemical Education
Figure 2. Karplus relationship. CIaphical representation of the original Karplus couDlina eomtion showin0 the anoular variation of lhe thre,bard . constant 3&. Thegraph iscomp&d ham the failowing relationship:3 & ~= 8.5 cos2d 0.28 when 0' d 90' and 9.5 cos2 d 0.28 when 90" < 4 > 180.
-
-
-
< >
-
Figure 3. Proton NMR spechum of cobra neurotoxin. The slgnafs from the protons are broader and occupy a larger chemical shin range lhan in the individualamino acids. Thls occurs becausethe protein rotates mare slowly in SolUtion than the small amino acid. Slower overall molecuisr motion results In broadenin0 of NMR line widths and affects the rate at which excited Drotans - ~~-~ refurn lo the nonexcited state. This nlormal8on can be dset.1 in studies of mo eculsr mollon and dynamics of protelns and DkA Tne Spacnum was recorded on a $aria" 400-MHz NMR at 23 'C. Minimal rero "ton enhancement has been applied
-
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~~
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on a laree coupline constant dataset.. dnn is the oroton. .--proton t&ionh aLgle, and the terms 6 and Xi desciibe the effect of orientation and substituent electroneeativitv. Forand tunately, the program PSEUROT written by & ~ e e i w Altona is available throueh the Quantum Chemistry Program Exchange and will perform the calrulationn automatically (4J.Analvsisof theproeramoutput provides the best fit betkeen suga; conformation and the experimentally determined proton-proton three-bond couplings. Theoretically, one codd deteimine the conformation of the sugar-phosphate backbone of DNA and RNA oligomers by analysis of the couoline network and maenitude of the couoline con. stant. In reality, this is not possible for even short oligomers. The broad resonance lines and comolexitv of DNA and protein spectra often prevent simple determination of the coupling constant. A spectrum of cobra neurotoxin-a 71amino-acid protein-is shown in Figure 3 to illustrate this point. The snectrum was recorded a t 23 OC on a 400-MHz spectromete;. Very few of the individual couplings can be discerned due to both the complexity of the spectrum and the increased linewidths. Comparison of the spectrum of tryptophan with cobra neurotoxin further illustrates these
.
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points. Generally, as molecules become larger they rotate more slowly in solution affecting the relaxation rate of the protons. This problem can be partially overcome by acquiring the data at higher temperatures (45 "C) and by the application of resolution enhancement procedures that sharpen the lines. Indeed, proton assignments and structural analysis of a-bunearotoxin, a similar cobra neurotoxin of 74 amino acids, wasieceutly reported 6 6 ) . The relaxation of nuclei back to equilibrium after excitation with a radiofrequency pulse occurs exponentially. The rate is characterized by two major relaxation mechanisms characterized by the time constants T I and Tz (7).The T I relaxation time describes the rate at which excited nuclei return to thermodynamic equilibrium ( 8 ) . The width of spectral lines is proportional to l / T z . The uncertainty principle dictates that if the T Zrelaxation rate is very fast, the lines will be broad. Slowly rotating macromolecules have rapid T z relaxation. Solid samples can be taken as an extreme example where T 2relaxation times are on the order of microseconds and line widths of kilohertz are common. The net result of this and other factors, such a s increases in solvent viscosity, is to increase the spectral line width in NMR spectra of biomolecules. Various dynamic processes including molecular motion and chemical exchange will also affect the relaxation, and processes with rates from approximately to s directly influence T I and T z (8). The maior problem in the analvsis of the NMR spectra of biologicalmoiecules is the compiexity of the spectra. Even short olipconucleotides and peptides often contain overlapping signals. In order to c&&mvent these problems and analyze the spectra of peptides and oligonucleotides, twodimensional spectroscopy is used. -
Two-Dlmenslonal NMR Spectroscopy (2D NMR)
A laree varietv of 2D NMR techniaues are currentlv aoplied inthe soluiion studies of macro~olecules.As the name of the technique implies, 2D experiments yield data along two frequency axes instead of one. The data produced from a 2D spectrum are often displayed as a contour plot in which the intensities of the resonancesare indicated hy the numher of contour levels.The presentationof thedata is thussimilar to a conventional contour map showine eeoloeical features. where tncreasing contour levels denote increasing height. A two-dimensional NMR sDectrum actuallv consists of a series of one-dimensional spec&a"stacked" ou"top of each other. A "stacked plot" of a two-dimensional spectrum of the amino acid valine showing each of the component spectra that compose the 2D plot is shown in Fimre 4. Contrast this with acontour plot ofthe same spectrumin Figure 5. The contour plot displays the data as if one were examinine the spectrum &om abovk. A one-dimensional spectrum o f t h e sample is plotted above and to the side of the 2D contour plot to aid in signal assignment. The two-dimensional spectrum consists of a diagonal representing protons that did not cross correlate during the experiment. The frequency of a peak on the diagonal is the same in both frequency dimensions (F1and Fz). The diagonal position of the u proton, for example, occurs a t 3.6 ppm in both F, and Fz. Coupling between Drotons is indicated bv off-diaeonal cross peaks. C o u ~ l i n e between the a and B protons is rndicated b i t w o cross peak; svmmetricallv disposed off the diagonal. The position of the cioss peak is diffeient with regard t o each frequency dimension. One cross peak occurs at 2.26 ppm on F , and 3.6 ppm on F2 while the other cross peak occurs at 3.6 ppm on I.', and 2.26 ppm on F2. The symmetrical disposition of cross peaks aids in assignment of spectra since each coupled proton pair can be connected by a boxdrawn between the diagonal peaks and the cross peaks connecting the protons. This is illustrated in Figure 5 by the box drawn between the 0 proton and the protons of the methyl groups of valine. The spectra in Figures 4 and 5 are COSY spectra. COSY is
--
I
,
,
,
,
3.5
3.0
, 2.5
2.0
,
,
1.5
2.0 PPW
Flgure 4. COSY staoked plot ot valine. Each of me individual spectra that make UD a twodimensional data set are shown. CH.
u
Figure 5. COSY wntour plot of vallne. The same 2D spechum in Figure 4 showing a contour plot where increasing contour levels mean increasing height. The diagonal and ofldlaganal cross peaks are Indicated. Volume 66
Number 2
February 1989
113
an acronym for correlated spectroscopy (7). COSY spectra yield information on the coupling patterns within a molecule. Each amino acid in a peptide & sugar ring in an oligosystem of protons that are coupled to nucleotide has a spin each other. Delineation of the spin systems in peptides and oligonucleotides permits identification of individual amino acids and sugars. However, COSY spectra do not provide information on the distances between individual protons. Likewise, COSY spectra do not provide data regarding which base is attached to a sugar or which aromatic ring is connected to the pentide chain. Such information is crucial to determine the- shution conformation and sequence of oligonucleotides and proteins. To obtain distance information an additional NMR technique is used that takes advantage of the nucler Overhauser effect (NOE) (8). The nuclear Overhauser effect is observed as an increase or decrease in the intensity of a proton resonance by exchange of energy with a neighboring proton. Nuclear Overhauser effect spectroscopy does not refer t o the throughbond coupling discussed above but results from energy transfer between protons through space. The transfer of energy is distance dependent, and the effect is not significant beyond about 4 A. The distance dependence permits one to determine quantitatively the distances between protons in a macromolecule. In the best cases accuracies of +0.2 A have been reported (9). Two types of NOE spectra are commonly used to investigate biomolecular structure. NOE difference spectra are one-dimensional spectra in whichaproton in the molecule is selectively excited by irradiation at its resonance frequency. Energy transfer to neighboring protons evolves viatime- an-d distance-dependent processes as the excited protons return to their nonexcited state. The transfer of energy between protons gives rise to the NOE effect. Spectra in which the wrotons were excited are subtracted from snectra in which no excitation was applied providing a difference spectrum. This techniaue can ~ r o d u c everv accurate distance information (9). he disadvantages of ihe technique are that selective irradiation of a single peak in the crowded spectra of macromolecules is difficult and often impossible. In addition, irradiation of each of the large - number of proton signals is prohibitively time consuming. Two-dimensional NOE spectroscopy (NOESY) circumvents both problems though the distance data obtained is less accurate. The data from a two-dimensional NOESY spectrum are identical in presentation to the COSY experiment, i.e, the spectrum is usually displayed as a contour plot with two frequency dimensions. The difference is that the cross peaks that appear in a NOESY spectrum indicate through-space interactions rather than couwline . between protons through bonds. The intensity of the cross peak is proportional to the distance between the two protons eivine " " &to the cross peak. Integration of cross peak intensities allows one to determine the distance between protons. The buildup of intensity of a NOE cross peak is not linearly related to the distance but is related by the inverse sixth power of the distance (I/$).The simplest way to determine the distance between two protons is to compare the intensity of the NOE cross peak between the two protons with the intensity of an NOE cross peak from two protons that are a known distance apart. This relationship can be expressed quantitatively by the following equation (9): ~
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them serves as an excellent distance marker for the analysis of RNA andDNAfraements. Further. the distances between protons in aromatic rings of proteins are likewise explicit. It should be noted that the eauation as formulated above is not strictly true. The rate of NOE buildup is actually the distance-dependent vhenomenon not the intensitv of a single NOE, and meas&ements of the rate of NOE buildup would theoretically provide more accurate distance measurements. However, the practical problems encountered when trying to measure the evolution of NOE in a biomolecule are considerable. Several measurements of the NOE intensity a t different time points are required for an accurate rate determination, and, at short time periods, when the amount of NOE buildup is small, the signal-to-noise ratio of the spectra may not permit accurate determination of the NOE. Fortunately, the errors introduced in using the simplification above are not generally large compared to experimental errors (9). Also, whether using NOE intensity or the rate of NOE buildup to determine distance, assumptions are often made about the relaxation of the protons, which may adversely affect the accuracy of the distance determination (9). Solutlon Structure Determlnatlon by NMR ~
~
Short, Double-Helical DNA Oligonucleotides
The three major conformational families of DNA (A, B, and Z form) can be readily distinguished by using 2D NMR. Each conformation of DNA is characterized by two conformational properties-the sugar pucker and the glycosidic bond angle. Five-membered rings are not flat but adopt envelope and twist shapes referred to as puckered forms. The envelope form denotes a conformation in which four of the atoms of the ring are in plane and the fifth atom lies above or below the plane. Twist forms have two of the atoms displaced above and below aplane formed by the other three atoms. In addition. the base mav adopt two orientations. svn and anti, relative the sugar birota'tion about the glycdsi"dic bond (I). Figure 6 illustrates sugar puckers and the syn and anti conformations. Syn conformation means the base points toward the sugar, while anti means the base is pointing away from the sugar(1). Changes in conformation;esult in different spatial relationships between the protons of the base and sugar. These differences in the spatial relationships of protons can be used t o characterize the conformation of short olieomers via NMR analvsis. eon experiments permit one to determine the nucleotide seauence of short olieomers. The occurrence of NOE's in a DNA chain is direcconal. Using the pattern of NOE contacts one can "sequence" a DNA oligomer, i.e., determine
ENVELOPE
TWIST
ry = r,,(N,IIN;j)'16
The term rij is an unknown distance between two protons desienated i and i. A known reference distance (. r....d denotes the &stance between the protons k and I , Nal is the magnitudeof theobserved NOE between wrotons kand /.and N.. is the magnitude of the NOE between protons i and'j (9). .' For example, the protons on the pyrimidine ring of cytosine (Hs and He) are fixed at 2.46 A, and the NOE between 114
Journal of Chemical Education
Figure 6. Envelope and twist conformtlons of fivemembered rings. Syn and anti wnformations of deoxyadenosine.
the order of bases. The H 1 sugar protons in A and B form DNA are approximately equidistant (-4.0 A) from their own base H8 (if the hase is a purine) or H6 (if the base is a pyrimidine) protons and thk H8 o; HI5 protons of the 5' (hut not 3') nucleotide. The assienmeut of the helix beeins at the 5' nucleotide because ever;nucleotide except theone at the Vend will have two NOE cross peaks. In right-handed DNA helices the sugar H I , ~Z~/HZ'protons g&e NOE's to the base H8 or H6 protons of the 3' neiehborine base but not to the 5' neighhoiing hase. In contrast, NOE'S occur between thymidine methyl protons and the H6/H8 protons of the 5' residue but not the 3' residue. After assignment of the base sequence the conformation of DNA may be determined. The pattern of NOE intensities is different in A form compared t o B form DNA (10). Aform DNA is characterized hv C3' endo suear pucker. and the intramolecular H6 or H8 HZ'distancc(3.8 A) is large compared to the internucleoe true for Btidedistances (1.7 A) (Fie. 7 ) (10).T h e o ~ o o s i t is form DNA. ~iffere&s & t h e ikensitiesbetween the interand intranucleotide NOE contacts between HZ' and H8 or
B FORM DNA C 2' END0 SUGAR PUCKER
A FORM DNA C 3'ENDO SUGAR PUCKER Figure 7. Dlagram showing the oonformatlons of the sugars and relatlve pOSitlonsof the bases in A- and B-form DNA. The relevent protons on the sugar and bases are numbered, and the distances betweenthe protons are indicated in angstroms (taken from Haasnoot et al. In Bform DNA, the number-2 carbon Is above the plane of the ring and theconformation Is referred to as C 2' endo. Whereas in Aform DNA it Is the number-3 oarbon that is above the plane of the rlng and this is referred to as C 3' enda.
(lo).
H6 base protons provide a sensitive method of discrimination between A- and B-form helices. Sequences with alternating purines and pyrimidines can adopt a left-handed helix, termed Z-form DNA. The ~ ..v r i m i dines have an anti configuration and C2'endo sugar pucker. The purines have a syn el~cosidichond and C3' endo suear pucker. The observed of NOE contacts thus dif& markedly from that observed for the right-handed helices and can be easily detected by NMR. Insolution, oligonucleotides in the Z form are in equilibrium with the B form. The B-to-Z transition can be induced in short olieonuclentides v ~ ~ with alternating pyrimidinepurine sequences by either high salt concentration or the addition of methanol (11,12). Under these conditions the exchange between the B and Z forms of DNA is slow and two separate sets of signals, one set from the B form and one set from the Z form, will be ohserved. The 2D NOE experiment can also detect slow exchange between species. dross peaks will appear between the protons of the B form and the corresponding protonsof the Z form. For example, in the sequence ~S'(GCGCGC)Zthe 5' guanine H8 proton in the B form will show a cross peak with the corresponding 5' guanine H8 proton in the Z form. Therefore, assignment of the protons of the oligonucleotide in the B form will by default assign the identical protons of the Z form (8, 17). While this method of assignment works well, alternative assienment schemes are possible that do not depend on exchange between B and Z fbrms (13,14). NMR studies on DNA are not limited to determination of which form of DNA the oligonucleotide adopts. Studies of the drug-DNA interactions and protein-DNA interactions are increasingly important areas of study. NMR can yield important information on the structure of drug-nucleic acid complexes and on the changes in conformation and stability of the DNA helix that occur upon drug binding (15). ~
~
~
In proteins and peptides sequential assignments are also accomplished by a combination of 21) NMR techniques. The assignment problem in peptides is more involved tban in DNA because there are 20 common amino acids and protein conformations are more varied tban DNA conformations. The variety of structural features in proteins ofwn requires the use of several other N M H experiments in addition to NOESY and COSY exnerimenm. The spin systems of amino acids can be determined using COSY spectra. COSY experiments on peptides do not provide sequence information. Again, as in DNA, NOESY experiments are used to obtain sequence information. The protons that are most critical to sequence determination in peptides are the protons on nitrogen atoms in the peptide backbone, Protons bonded to nitrogen exchange with DzO, and experiments must be performed in HpO solution. Performing proton NMR experiments in water, however, presents a particular problem because the protons in water ahsorb radiation a i d the concentration of water is much greater than the concentration of the macromolecule in the sample, which is typically 1-5 mm. Many signals from the macromolecule are thus obscured by the water signal. In addition a further problem is encountered; the high dynamic range of the signals in the sample. A major problem arises in FT-NMR when small signals must be detected in the presence of a very large solvent signal. There is a finite limit on the NMR instrument's ability to digitize signals (i.e., convert the analog output of the receiver to digital data the computer can use) (16). The net result is that small signals may not be properly digitized and can become lost in the random noise of a very large peak. Thus, the intensity of the water signal must be decreased (suppressed) to observe the sample's signals. There are several methods of water suppression currently available ( 9 ) .Most involve selective excitation of the orotons in the sample in sucha way that only signals from thksample Volume 66
Number 2
February 1969
115
~
~
Figure 8. intenelatlonshlps ol protons in a peptide linkage. The experiments useful for assignments of the sequence of amlna acids in a peptide are indicated. Pathway A depicts the connection between the fi proton of the amino acid with the NH proton of that amino acid. This information can be determined from a RELAY experiment. A COSY experiment in H20 can be usedto connectthe NH withthe a proton (pathway establishing the spin can be determined set for the amino acid. Finsik, the seouence ol the .motide . ov using NOESY data (pathwiys C an; 0 ) The o proton of the am no acoo u I $now an NO€ to tne NH proton of the aojecent base (pathway C I . Pathway 0 snows connection between the NH ol one amino acid and m e hd of the nsn.
thus
and not the water resonance are excited. Since no direct excitation is a ~ n l i e dto the water resonance. the intensitv of the water sign; is effectively suppressed. ' Observation of the protons on nitrogen in aqueous samples permits sequential assignment of the peptide backbone. In principle, the a protons can he connected to the B protons via COSY spectra. The first assignments of were performed using only COSY and NOE data; however, the large amount of overlap between a protons in large peptides makes the assignment difficult (8). A second type of 2D NMR exneriment. the RELAY experiment. is extensivelv used in assignment of peprides to circumvent this problem. In the RELAY exneriment cross neaks occur not onlv between directly coupled protons b i t also between indirectly coupled protons. In the peptide backbone, the proton on nitrogen will couple to the a proton and the a protons will couple t o the B protons. Therefore, in such an analysis the twosets of COSY cross peaks will occur between the NH and u protons and between the a and B protons. In the RELAY experiment an additional cross between the 9 , protons and the distant NH proton will occur (8).This experiment permits direct assignment of the side chains of amino acids to the NH of a peptide bond even if the a protons are obscured. Seauential assienment of the amino acids can then be made using these datain combination with NOESY data. The a proton of the amino acid will show an NOE to the NH proton of the next residue. Additional NOE's may he ohserved between the B Drotons of one amino acid and the NH of the adjacent am& acid as well as between the two NH protons. Figure 8 illustrates the COSY, RELAY, and NOE connectivities expected between adjacent amino acids. Using this information, sequences of amino-acids can he confirmed and compared with the sequence of the peptide thus establishing the position of the amino acids in the peptide. Assignment techniques have progressed to the point that NMR data can be used to determinate the amino acid sequence of a peptide and NMR data has been used to correct reported amino acid sequences (17,18). The different folding patterns of peptides will cause amino acids separated from each other in sequence to he close to each other in space. NOE data will help establish the proximity of these amino acids in space. Information on the ~roximitvof amino acids in space combined with seauence and coupling information can he combined t'o provide an overall three-dimensional structure of the peptide. Computer Modeling and NMR Analysis Model building has been very important t o our understanding of macromolecular structure. The complexity of 116
Journal of Chemical Education
I I
II I
I
Flgure 9. Computer graphics image of B-form DNA. he image was produced an an IBM PC running in Tektronix 4107 terminal emulation made to a Digital Equipment Corporation VAX 785 computer.
macromolecular structure makes visualization of the structures difficult. C o m ~ u t e rmodeline of macromolecular structure using compiter graphics techniques has become an indispensable aid in the determination of macromolecular strufture. Several computer graphics systems are available for modeling protein and DKA structures. The systems vary greatly with;egard t o the quality (and price!jof the graphical display. Figure 9 shows a "low-end" computergraphics image of B-form DNA. This image was constructed using Still's program MacroModel running on a VAX 785 comnuter. The imaee was dis~lavedon an IBM nersonal computer running siftware that emulates a ~ e k t r d n i x4107 graphics terminal. While the graphics quality is not comparable to a high-resolution graphics terminal, the display is certainlv of sufficient aualitv . . to be verv useful for analvsis of the str&ture and the software is inexpensive and readily available. Computer graphics is only one part of the procedure for understanding the three-dimensional structure of macromolecules. Most currently available software packages are integrated graphics and computational packages containing molecular mechanics programs. Molecular mechanics calculations permit the determination of the minimum energy structures of macromolecules. One such program widely used for energy calculations on macromolecules is AMBER (19). Most software packages permit inclusion of distance constraints obtained from NOE data directly into the calculation. Therefore, one can use the data obtained on proton distances from NMR to constrain certain protons in the macromolecule to he a fixed distance (actually a distance range) apart. This constrains the protein or DNA so that the calculations cannot move those protons significantly from the experimentally determined values hut can adjust the structure to the lowest energy (and hence most probable) form. One problem with this method of analysis is that it basically represents the macromolecule as a static structure. In solution, molecules and their constituent atoms are in constant motion. Therefore, i t has been argued that a better method of modeline macromolecular svstems is to use restrained molecular ldynamics calculati&s (20). Restraihed molecular dynamics calculations permit one to simulate molecular motion inmacromolecules and torefine the structure of the molecule to match the experimentally determined
NMR data. Dataon the distances between protonsobtained from careful NOE ex~erimentsare entered into the computer, and calculations are performed to refine the structure. The great advantage to this analysis technique is that the data from NOE contacts are solution data and they reflect the averaee closest contact between two protons. The resultant structurecan then beviewed in threedimensionsonthe computer-graphics screen and can be rotated and expanded to i&esti&t&individual interactions. Research on the best procedur& for the incorporation of NOE data into molecular mechanics calculations is an active area of interest. There is currently no widely accepted method for the integration of experimental and theoretical data nor is there a generally accepted molecular mechanics force field. While AMBER is extensively utilized, vida supra, a variety of other programs are available including CHARM, OPLSA, and several commercial software systems (21,22). Conclusions
NMR analysis can provide detailed information on the solution conformation of oligonucleotides and small proteins. Like all analvtical techniaues. NMR analvsis has limited applicability. kt the presen't time, detailedUinformation can only be obtained for small oligonucleotides and proteins with molecular weights of 15,000 daltons or less (6). This certainly does not preclude NMR studies of larger systems, but the quality of data decreases with increasing molecular size. A second limitation is that NMR is a relatively insensitive analytical technique. Even the most powerful instruments require millimolar concentrations of sample. Therefore, probiems related to self-association of the macromolecules are common. Despite the limitations, NMR analyais has become a common tool to investigate the solution structure of small macromolecules. The uniaue feature of the technioue is that information aboutthe solution structure of the mb~eculecan he obtained. NMR also provides an excellent method to
exnlore the dvnamics of nroteins and DNA. The future for examination of nucleic acids and proteins by NMR is bright. New 2D NMR techniques are constantly being introduced that simplify the analysis of complex NMR spectra. Indeed, recently a technique for performing threedimensional (3D) NMR experiments was introduced to aid in the analvsis of nrotein structures (16). Novel experimental techniques ~ o ; ~ l e dwith more powerful comnuters and hieher field instruments will undoubtedly expand&theapplicab&ty of the technique in the future. ~~~
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~~
Literature Clted
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