Solution conformation of thymosin beta 4: a nuclear magnetic resonance and simulated annealing study

The conformation of the polypeptide thymosin beta 4 in solutions of 60% (v/v) trifluoroethanol-d3 and 50% (v/v) hexafluoroisopropyl-d2 alcohol in water is investigated by nuclear magnetic resonance (NMR) spectroscopy. Under these conditions thymosin beta 4 adopts an ordered structure. By use of a combination of two-dimensional NMR techniques, the 1H NMR spectrum of thymosin beta 4 is assigned. A set of 180 approximate interproton distance constraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 33 phi constraints obtained for JNH alpha coupling data and the 23 psi dihedral angles identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Ten independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 30 calculated structures satisfy the experimental constraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 30 converged structures indicates that there are two helical regions extending from residues 4-16 and from residues 30-40, which are well defined both in terms of atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 2 A. The two helices exhibit typical amino acid preferences for specific locations at the ends of helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 15-23), and two extended strands (residues 24-31 and 34-40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.     

Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin. SCM is more stable than the native monellin for both heat and acidic environments. Data from gel filtration HPLC and NMR indicate that the SCM exists as a monomer in aqueous solution. The solution structure of SCM has been determined by nuclear magnetic resonance (NMR) spectroscopy and dynamical simulated annealing calculations. A stable alpha-helix spanning residues Phe11-Ile26 and an antiparallel beta-sheet formed by residues 2-5, 36-38, 41-47, 54-64, 69-75, and 83-88 have been identified. The sheet was well defined by backbone-backbone NOEs, and the corresponding beta-strands were further confirmed by hydrogen bond networks based on amide hydrogen exchange data. Strands beta2 and beta3 are connected by a small bulge comprising residues Ile38-Cys41. A total of 993 distance and 56 dihedral angle restraints were used for simulated annealing calculations. The final simulated annealing structures (k) converged well with a root-mean-square deviation (rmsd) between backbone atoms of 0.49 A for secondary structural regions and 0.70 A for backbone atoms excluding two loop regions. The average restraint energy-minimized (REM) structure exhibited root-mean-square deviations of 1.19 A for backbone atoms and 0.85 A for backbone atoms excluding two loop regions with respect to 20 k structures. The solution structure of SCM revealed that the long alpha-helix was folded into the concave side of a six-stranded antiparallel beta-sheet. The side chains of Tyr63 and Asp66 which are common to all sweet peptides showed an opposite orientation relative to H1 helix, and they were all solvent-exposed. Residues at the proposed dimeric interface in the X-ray structure were observed to be mostly solvent-exposed and demonstrated high degrees of flexibility.     

A proton nuclear magnetic resonance study of the conformation of bovine anaphylatoxin C5a in solution

The solution conformation of bovine anaphylatoxin C5a has been investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H-NMR spectrum is assigned in a sequential manner using a variety of two-dimensional NMR techniques. A qualitative interpretation of the short range nuclear Overhauser enhancement data involving the NH, C alpha H and C beta H protons suggests that C5a has four helices comprising residues 5-11, 15-25, 33-39 and 46-61, and is composed of a globular head (residues 5-61) and a C-terminal tail. The polypeptide fold was determined by hybrid distance geometry-dynamical simulated annealing calculations on the basis of 203 approximate interproton distance restraints, 22 distance restraints for 11 intrahelical hydrogen bonds (identified on the basis of the pattern of short range NOEs and slowly exchanging backbone amide protons) and restraints for the 3 disulfide bridges. The overall polypeptide fold is similar to that of the sequence related human recombinant anaphylatoxin C5a [(1988) Proteins 3, 139-145].     

Three-dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometry

The solution conformation of acyl carrier protein from Escherichia coli (77 residues) has been determined on the basis of 423 interproton-distance restraints and 32 hydrogen-bonding restraints derived from NMR measurements. A total of nine structures were computed using a hybrid approach combining metric matrix distance geometry and dynamic simulated annealing. The polypeptide fold is well defined with an average backbone atomic root-mean-square difference of 0.20 +/- 0.03 nm between the final nine converged structures and the mean structure obtained by averaging their coordinates. The principal structural motif is composed of three helices: 1 (residues 3-12), 2 (residues 37-47) and 4 (residues 65-75) which line a hydrophobic cavity. Helices 2 and 4 are approximately parallel to each other and anti-parallel at an angle of approximately equal to 150 degrees to helix 1. The smaller helix 3 (residues 56-63) is at an angle of approximately equal to 100 degrees to helix 4.     

Determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing

The complete three-dimensional structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima in aqueous solution was determined on the basis of 324 interproton distance constraints, 80 non-nuclear Overhauser effect distances, and 22 hydrogen-bonding constraints, supplemented by 27 phi backbone angle constraints derived from nuclear magnetic resonance measurements. The nuclear magnetic resonance input data were converted to the distance constraints in a semiquantitative manner after a sequence specific assignment of 1H spectra was obtained using two-dimensional nuclear magnetic resonance techniques. Stereospecific assignments were obtained for 17 of the 48 prochiral centers of the squash trypsin inhibitor using the floating chirality assignment introduced at the dynamical simulated annealing stage of the calculations. A total of 34 structures calculated by a hybrid distance geometry-dynamical simulated annealing method exhibit well-defined positions for both backbone and side-chain atoms. The average atomic root-mean-square difference between the individual structures and the minimized mean structure is 0.35(+/- 0.08) A for the backbone atoms and 0.89(+/- 0.17) A for all heavy atoms. The precision of the structure determination is discussed and correlated to the experimental input data.     

Two-dimensional nuclear magnetic resonance study of brain natriuretic peptide in aqueous solution.

Two dimensional NMR spectra of porcine brain natriuretic peptide have been recorded at 400 MHz. Peak assignments have been made and the combined information from chemical shifts, coupling constants, temperature coefficients, exchange studies and nuclear Overhauser effects has been used to determine the conformation of pBNP in aqueous media. Overall the peptide appears to be conformationally averaged with the possibility of some restricted flexibility in localized regions. The conformation of porcine brain natriuretic peptide in water is compared to previous studies in d6-DMSO and to studies of atrial natriuretic peptide and some closely related analogues in H2O and d6-DMSO.     

Solution structure of recombinant hirudin and the Lys-47----Glu mutant: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study

The solution structure of recombinant wild-type hirudin and of the putative active site mutant Lys-47----Glu has been investigated by nuclear magnetic resonance (NMR) spectroscopy at 600 MHz. The 1H NMR spectra of the two hirudin variants are assigned in a sequential manner with a combination of two-dimensional NMR techniques. Some assignments made in our previous paper [Sukumaran, D. K., Clore, G. M., Preuss, A., Zarbock, J., & Gronenborn, A. M. (1987) Biochemistry 26, 333-338] were found to be incorrect and are now corrected. Analysis of the NOE data indicates that hirudin consists of an N-terminal compact domain (residues 1-49) held together by three disulfide linkages and a disordered C-terminal tail (residues 50-65) which does not fold back on the rest of the protein. This last observation corrects conclusions drawn by us previously on hirudin extracted from its natural source, the leech Hirudo medicinalis. The improved sensitivity of the 600-MHz spectrometer relative to that of our old 500-MHz spectrometer, the availability of two variants with slightly different chemical shifts, and the additional information arising from stereospecific assignments of methylene beta-protons and methyl protons of valine have permitted the determination of the solution structure of hirudin with much greater precision than before. Structure calculations on the N-terminal domain using the hybrid distance geometry-dynamical simulated annealing method were based on 685 and 661 approximate interproton distance restraints derived from nuclear Overhauser enhancement (NOE) data for the wild-type and mutant hirudin, respectively, together with 16 distance restraints for 8 backbone hydrogen bonds identified on the basis of NOE and amide NH exchange data and 26 phi backbone and 18 chi 1 side-chain torsion angle restraints derived from NOE and three-bond coupling constant data. A total of 32 structures were computed for both the wild-type and mutant hirudin. The structure of residues 2-30 and 37-48 which form the core of the N-terminal domain is well determined in both cases with an average atomic rms difference between the individual structures and the respective mean structures of approximately 0.7 A for the backbone atoms and approximately 1 A for all atoms. As found previously, the orientation of the exposed finger of antiparallel beta-sheet (residues 31-36) with respect to the core could not be determined on the basis of the present data due to the absence of any long-range NOEs between the exposed finger and the core.(ABSTRACT TRUNCATED AT 250 WORDS)     

A proton nuclear magnetic resonance study on the solution structure of crotamine

Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. ThepH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidicpH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for He-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.     

Lipopolysaccharide‐bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad‐spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram‐negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram‐negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α‐helical structure in a solution containing LPS. For NMR experiments, we expressed ¹⁵N‐labeled and ¹³C‐labeled CP1 in bacterial cells and successfully assigned almost all backbone and side‐chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr‐NOE) experiments in LPS. We performed ¹⁵N‐edited and ¹³C‐edited Tr‐NOE spectroscopy for CP1 bound to LPS. Tr‐NOE peaks were observed at the only C‐terminal region of CP1 in LPS. The results of structure calculation indicated that the C‐terminal region (Lys15–Gly29) formed the well‐defined α‐helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proton nuclear magnetic resonance study of the third component of complement: solution conformation of the carboxyl-terminal segment of C3a fragment

A proton nuclear magnetic resonance (NMR) study is reported of des-Arg-C3a, which is a 76-residue fragment obtained from the N-terminal portion of the alpha chain of the third component of human complement. A method of carboxypeptidase digestion/difference spectroscopy [Endo, S., & Arata, Y. (1985) Biochemistry 24, 1561-1568] was used for the spectral assignments for Ala-76, Leu-75, Gly-74, His-72, His-67, and Ala-48. On the basis of the NMR results obtained for these residues, we conclude that in aqueous solution (1) the C-terminal segment Leu-73-Ala-76 is free from interactions with the rest of the C3a molecule and (2) the major part of the C-terminal segment takes an ordered conformation. We also suggest that the presence of a core, which is formed by segment Tyr-15-Tyr-59 [Huber, R., Scholze, H., Paques, E. P., & Deisenhofer, J. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1389-1399], is essential for the C-terminal segment in maintaining the ordered structure in aqueous solution. 1H NMR spectral data were also obtained for the intact C3 from human and porcine sources. The resonances for the C2-H protons of His-67 and His-72, which exist in the C3a part of the human C3 molecule, were assigned. Comparisons of the results obtained with those for des-Arg-C3a demonstrate that upon cleavage of C3a very little change, if any, is induced in microenvironments of His-67 and His-72 and a piece of segment that contains His-72 is exposed to solvent and highly flexible.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-terminal half of a mitochondrial presequence peptide takes a helical conformation when bound to dodecylphosphocholine micelles: a proton nuclear magnetic resonance study

Two-dimensional proton nuclear magnetic resonance (NMR) spectra of a synthetic peptide (p25) corresponding to the amino-terminus of the yeast mitochondrial cytochrome oxidase subunit IV precursor protein have been analyzed. Sequence-specific resonance assignments of the peptide have been made in the presence of micelles of a phospholipid analog, perdeuterated dodecylphosphocholine (DPC), with the aid of such techniques as HOHAHA, DQF-COSY, and NOESY. The interresidue nuclear Overhauser effects (NOEs) indicate that the N-terminal half of p25 (S3-F11) takes a helical structure while the C-terminal half does not take a regular secondary structure. Addition of DPC to the solution of p25 induced chemical shift changes only of the resonances from the residues in the N-terminal half, suggesting that the N-terminal half of p25 is directly involved in binding to DPC. The induced helical structure in the N-terminal half at a lipid-water interface may be important in the ability of this presequence to direct a "passenger" protein into mitochondria.     

Conformational study of a custom antibacterial peptide cecropin B1: implications of the lytic activity

Cecropin B1 (CB1) with two amphipathic alpha-helical segments is a derivative of the natural antibacterial peptide, cecropin B. The assays of cell lysis show that, compared with cecropin A (CA), CB1 has a similar ability to lyse bacteria with a higher potency (two- to six-fold higher) in killing cancer cells. The difference may be due to the fact that the peptides possess different structures and sequences. In this study, the solution structure of CB1 in 20% hexafluoroisopropanol was determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The (1)H NMR resonances were assigned. A total of 350 inter-proton distances were used to calculate the solution structure of CB1. The final ensemble structures were well converged, showing the minimum root mean square deviation. The results indicate that CB1 has two stretches of helices spanning from residues 3 to 22 and from residues 26 to 33, which are connected by a hinge section formed by Gly-23 and Pro-24. Lys-25 is partially incorporated in the hinge region. The bent angle between two helical segments located in two planes was between 100 and 110 degrees. With comparisons of the known NMR structure of CA and its activities on bacteria and cancer cells, the structure-function relationship of the peptides is discussed.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

A receptor-binding peptide from human interleukin-6: isolation and a proton nuclear magnetic resonance study.

In order to locate the receptor-binding region of human interleukin-6 (IL-6), twelve peptide fragments were prepared by digestion of IL-6 with lysylendopeptidase. A significant activity of the receptor-binding was observed only for a peptide Ile88-Lys121, although the activity was estimated at 10(4)-fold less than that of intact IL-6. Solution structure of the peptide Ile88-Lys121 was analyzed by using two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The results indicate the presence of alpha-helices in the regions Leu93-Phe106 and Glu110-Ser119. On the basis of the NMR data, we also prepared two peptides. Four-fold less binding activity than that of the peptide Ile88-Lys121 was observed for the peptide Ile88-Arg105, but no activity for the peptide Glu110-Lys121. These results suggest that the helical peptide Ile88-Arg105 composes a part of the receptor-binding region.     

The structure of the mammalian antibacterial peptide cecropin P1 in solution, determined by proton-NMR.

Cecropins are peptides with antibacterial activity originally found in insects. Recently a cecropin-type peptide was isolated from pig intestine. This peptide, porcine cecropin P1, which has 31 amino acid residues and is not amidated in the C-terminus, has been synthesized, purified, and investigated by CD and two-dimensional 1H-NMR at pH 5.0 in aqueous solution with 30% (by vol.) 1,1,1,3,3,3-hexafluoro-2-propanol. All proton resonances have been assigned except for the N-terminal serine. Using constraints derived from NOE connectivities and 3JNH alpha-coupling constants, three-dimensional structures have been calculated by means of a distance-geometry program. Some of these structures have been refined by energy minimization and restrained molecular dynamics. The structures reveal an alpha-helix of approximately seven turns along nearly the full length of the peptide. The central part of the helix is very well defined by the NMR constraints. Also the chemical shifts of the alpha protons and the results of CD measurements are in accord with this structure, which is different from the helix-hinge-helix structure earlier found in cecropin A and related peptides. In the alpha-helix of cecropin P1 there is a long amphipathic section, of 4-5 turns, and a short hydrophobic section of one to two turns, with an intervening Glu-Gly sequence, which is a potential bend-forming section. The helix can easily span a lipid membrane.     

Nuclear magnetic resonance studies and molecular dynamics simulations of the solution conformation of a 'designed', alpha-helical peptide.

The complete three-dimensional structure in methanol of an amphipathic alpha-helical peptide, that has been designed by taking into account the three-dimensional structures of small haemolytic peptides, secondary structure prediction algorithms and the well documented literature on alpha-helix stabilizing factors, has been elucidated by two-dimensional NMR spectroscopy. Initially various two-dimensional spectra (COSY, TOCSY, and NOESY) allowed the complete sequence specific assignment of all signals in the 1H spectrum. Consequently trial structures were generated which were then subjected to molecular dynamics simulations using 121 NOE-derived distances and 25 vicinal coupling constant values as structural restraints to give a final set of calculated structures. These structures are in complete agreement with the results of a circular dichroism study and reveal that the peptide adopted a highly ordered alpha-helical conformation. Details of the structure which throw light on future peptide/protein design are discussed.     

A nuclear magnetic resonance study of the hydrogen-exchange behaviour of lysozyme in crystals and solution

Amide hydrogen/deuterium exchange behaviour has been studied for all of the peptide amides of hen lysozyme by means of two-dimensional n.m.r. spectroscopy. The amides have been grouped into four categories on the basis of their rates of exchange in solution at pH 4.2 and 7.5. The distribution of the amides into the different categories has been examined in the light of the crystallographic structural information, considering the type of secondary structure, the nature of hydrogen bonding and the distance from the protein surface. None of these features was found to determine uniquely the pattern of hydrogen exchange rates within the protein. The exchange behaviour of the individual amides could, however, in general be rationalized by a combination of these features. Hydrogen exchange was also monitored in both tetragonal and triclinic crystals of lysozyme, by allowing exchange to take place in the crystals prior to dissolution and recording of n.m.r. spectra under conditions where further exchange was minimized. This enabled direct comparison to be made of the exchange behaviour in the crystals and solution. A reduction in exchange rate was observed in the crystalline state relative to solution for a substantial number of amides and distinct differences between exchange in the different crystals could be observed. These differences between the solution and the different crystal states do not, however, correlate in a simple manner with proximity to intermolecular contacts in the crystals. However, the existence of these contacts, which are on the surface of the protein molecule, have a profound effect on the exchange of amides in the interior of the protein. The results indicate that the spectrum of fluctuations giving rise to hydrogen exchange may be significantly altered by the intermolecular interactions present within the crystalline state.     

Ribosome-chloramphenicol interactions: a nuclear magnetic resonance study

Proton magnetic resonance line broadening of chloramphenicol resonances has been employed to study the binding of this inhibitor of protein synthesis to the Escherichia coli 70 S ribosome. Temperature dependence measurements of the resonance line widths show that chloramphenicol is in fast exchange with the ribosome. Differential broadening of the various drug resonances suggests that it binds in its receptor site in essentially the same conformation that exists free in solution. Thus, even though the drug possesses a fair degree of structural flexibility, this is not necessary for its interaction with the ribosome. The recognition is most likely of the classic lock and key type, with the ribosomal site being essentially an open gate for the fitting of the drug. From the proton line-width measurements and ¹⁹F spectra of a derivative, it has been possible to propose a model for the geometry of chloramphenicol when it resides on the ribosome which is consistent with established structure-activity relationships for the drug.