Horizontal head impulse test detects gentamicin vestibulotoxicity.

BACKGROUND: Parenteral antibiotic therapy with gentamicin, even in accepted therapeutic doses, can occasionally cause bilateral vestibular loss (BVL) due to hair cell toxicity. OBJECTIVE: To quantify in patients with gentamicin vestibulotoxicity (GVT) the extent of acceleration gain deficit of the horizontal vestibulo-ocular reflex at different accelerations with a graded head impulse test (HIT) in comparison with standard caloric and rotational testing. To characterize the corresponding HIT catch-up saccade pattern to provide the basis for its salience to clinicians. METHODS: Horizontal HIT of graded acceleration (750 degrees-6,000 degrees/sec2) was measured with binocular dual search coils in 14 patients with GVT and compared with 14 normal subjects and a control subject with total surgical BVL. RESULTS: Patients showed mostly symmetric HIT gain deficits with a continuous spectrum from almost normal to complete BVL. Gain deficits were present even at the lowest head accelerations. HIT gain correlated better with caloric (Spearman rho = 0.85, p = 0.0001) than rotational testing (rho = 0.55, p = 0.046). Cumulative amplitude of overt saccades after head impulses was 5.6 times larger in patients than in normal subjects. Compared with previously published patients after unilateral vestibular deafferentation, GVT patients with BVL generated only approximately half the percentage of covert saccades during head rotation (23% at 750 degrees/sec2 to 46% at 6,000 degrees/sec2). CONCLUSIONS: Head impulse testing is useful for early bedside detection of gentamicin vestibulotoxicity because most patients, even those with partial bilateral vestibular loss (BVL), have large overt saccades. Covert saccades, which can conceal the extent of BVL, are only approximately half as frequent as in unilateral patients, but may be present even in total BVL.

Gentamicin vestibulotoxicity impairs human electrically evoked vestibulo-ocular reflex.

BACKGROUND: Electrical vestibular stimulation is believed to directly activate the vestibular afferents to mediate an electrically evoked vestibulo-ocular reflex (eVOR). Gentamicin, an aminoglycoside antibiotic, induces vestibulotoxicity by hair cell damage and death. OBJECTIVE: To determine if human eVOR is impaired by hair cell damage and death in systemic gentamicin vestibulotoxicity (GV). METHODS: Three-dimensional binocular eye movements evoked by bilateral, bipolar, 100 msec direct current-step at intensities of 0.9, 2.5, 5.0, 7.5, and 10.0 mA were recorded with dual-search coils in 12 GV patients, and the results were compared to 13 healthy subjects. RESULTS: Normal eVOR was predominantly torsional, comprising phasic eVOR initiation and cessation acceleration pulses at 9 msec latency after current onset and offset, with a tonic eVOR velocity-step during the 100 msec intervening period of maintained current. Normal phasic eVOR increased, while tonic eVOR scaled linearly, with current intensity. GV impaired phasic eVOR more severely than tonic eVOR, and prolonged the latency to 12-13 msec. In patients without mechanical response to vestibular tests, phasic eVOR was reduced to one-fifth of normal amplitude, doubled in duration, had reduced ability to vary with current intensity, and threshold was increased. Tonic eVOR was reduced to one-third of normal, but still scaled linearly with current intensity. Patients, who retained partial mechanical responses to vestibular tests, had phasic eVOR impairment without tonic eVOR abnormality. CONCLUSION: Impairment of evoked vestibulo-ocular reflex (eVOR) in gentamicin vestibulotoxicity (GV) suggests that vestibular hair cells, activated by electrical stimulation, mediate the eVOR. Abnormalities of the eVOR, especially the phasic component, might be a marker of vestibular injury in GV.

Head impulse test in unilateral vestibular loss: vestibulo-ocular reflex and catch-up saccades.

BACKGROUND: Quantitative head impulse test (HIT) measures the gain of the angular vestibulo-ocular reflex (VOR) during head rotation as the ratio of eye to head acceleration. Bedside HIT identifies subsequent catch-up saccades after the head rotation as indirect signs of VOR deficit. OBJECTIVE: To determine the VOR deficit and catch-up saccade characteristics in unilateral vestibular disease in response to HIT of varying accelerations. METHODS: Eye and head rotations were measured with search coils during manually applied horizontal HITs of varying accelerations in patients after vestibular neuritis (VN, n = 13) and unilateral vestibular deafferentation (UVD, n = 15) compared to normal subjects (n = 12). RESULTS: Normal VOR gain was close to unity and symmetric over the entire head-acceleration range. Patients with VN and UVD showed VOR gain asymmetry, with larger ipsilesional than contralesional deficits. As accelerations increased from 750 to 6,000 degrees /sec(2), ipsilesional gains decreased from 0.59 to 0.29 in VN and from 0.47 to 0.13 in UVD producing increasing asymmetry. Initial catch-up saccades can occur during or after head rotation. Covert saccades during head rotation are most likely imperceptible, while overt saccades after head rotation are detectable by clinicians. With increasing acceleration, the amplitude of overt saccades in patients became larger; however, initial covert saccades also became increasingly common, occurring in up to about 70% of trials. CONCLUSIONS: Head impulse test (HIT) with high acceleration reveals vestibulo-ocular reflex deficits better and elicits larger overt catch-up saccades in unilateral vestibular patients. Covert saccades during head rotation, however, occur more frequently with higher acceleration and may be missed by clinicians. To avoid false-negative results, bedside HIT should be repeated to improve chances of detection.

Click-evoked vestibulo-ocular reflex: stimulus-response properties in superior canal dehiscence.

BACKGROUND: An enlarged, low-threshold click-evoked vestibulo-ocular reflex (VOR) can be averaged from the vertical electro-oculogram in a superior canal dehiscence (SCD), a temporal bone defect between the superior semicircular canal and middle cranial fossa. OBJECTIVE: To determine the origin and quantitative stimulus-response properties of the click-evoked VOR. METHODS: Three-dimensional, binocular eye movements evoked by air-conducted 100-microsecond clicks (110 dB normal hearing level, 145 dB sound pressure level, 2 Hz) were measured with dual-search coils in 11 healthy subjects and 19 patients with SCD confirmed by CT imaging. Thresholds were established by decrementing loudness from 110 dB to 70 dB in 10-dB steps. Eye rotation axis of click-evoked VOR computed by vector analysis was referenced to known semicircular canal planes. Response characteristics were investigated with regard to enhancement using trains of three to seven clicks with 1-millisecond interclick intervals, visual fixation, head orientation, click polarity, and stimulation frequency (2 to 15 Hz). RESULTS: In subjects and SCD patients, click-evoked VOR comprised upward, contraversive-torsional eye rotations with onset latency of approximately 9 milliseconds. Its eye rotation axis aligned with the superior canal axis, suggesting activation of superior canal receptors. In subjects, the amplitude was less than 0.01 degrees, and the magnitude was less than 3 degrees/second; in SCD, the amplitude was up to 60 times larger at 0.66 degrees, and its magnitude was between 5 and 92 degrees/second, with a threshold 10 to 40 dB below normal (110 dB). The click-evoked VOR magnitude was enhanced approximately 2.5 times with trains of five clicks but was unaffected by head orientation, visual fixation, click polarity, and stimulation frequency up to 10 Hz; it was also present on the surface electro-oculogram. CONCLUSION: In superior canal dehiscence, clicks evoked a high-magnitude, low-threshold, 9-millisecond-latency vestibulo-ocular reflex that aligns with the superior canal, suggesting superior canal receptor hypersensitivity to sound.

Benign positional nystagmus: a study of its three-dimensional spatio-temporal characteristics.

OBJECTIVE: To describe the spatial and temporal characteristics of benign positional nystagmus (BPN) subtypes in benign positional vertigo (BPV) due to vestibular lithiasis affecting one or more semicircular canals (SCCs). BACKGROUND: Activation of SCC receptors by sequestered otoconia, either freely moving (canalithiasis) or cupula-adherent (cupulolithiasis) during head position changes with respect to gravity, is the accepted cause of BPV. Although accurate identification and interpretation of BPN is critical to BPV therapy, no rigorous, kinematically correct three-dimensional spatio-temporal analysis of BPN in all its forms exists. METHODS: Using dual-search scleral coils, the authors recorded BPN provoked by Dix-Hallpike or supine ear-down test in a two-axis whole-body rotator in 44 patients with refractory BPV. To localize the SCC affected, BPN rotation axes were compared to SCC axes, axes orthogonal to average SCC planes. RESULTS: Sixteen patients had upbeat, geotropic-torsional BPN in the Dix-Hallpike test to one side and five to both sides, with BPN rotation axes clustered around the lowermost posterior SCC axis. Seven had direction-changing horizontal BPN, three geotropic (canalithiasis) and four apogeotropic (cupulolithiasis), with rotation axes around the lowermost and uppermost horizontal SCC axis. Seven had predominantly downbeating BPN with rotation axes clustered around one superior SCC axis. Nine had upbeat, horizontal-torsional BPN with rotation axes located between posterior and horizontal SCC axes of the lowermost ear suggesting simultaneous lithiasis in both SCCs. BPN vector-guided repositioning therapy was successful in 43 patients. CONCLUSION: Benign positional vertigo can affect one or more semicircular canals and three-dimensional recording with vector analysis of the benign positional nystagmus (BPN) can guide canalith repositioning therapy especially in refractory cases with atypical BPN.

The click-evoked vestibulo-ocular reflex in superior semicircular canal dehiscence.

The authors studied eye movement responses to loud (110dB) clicks in 4 patients with Tullio effect due to superior semicircular canal dehiscence and in 9 normal subjects, by averaging the electro-oculogram. All 4 patients had small (0.1-0.3 deg) but easily reproducible vertical vestibulo-ocular reflex eye movement responses to the clicks. Normal subjects had responses that were at least 10 times smaller. The click-evoked vestibulo-ocular reflex test is a simple, robust way to screen dizzy patients for symptomatic superior semicircular dehiscence.

Effects of unilateral vestibular deafferentation on the linear vestibulo-ocular reflex evoked by impulsive eccentric roll rotation.

The effects of unilateral vestibular deafferentation (UVD) on the linear vestibulo-ocular reflex (LVOR) were studied by measuring three-dimensional eye movements in seven UVD subjects evoked by impulsive eccentric roll rotation while viewing an earth-fixed target at 200, 300, or 600 mm and comparing their responses to 11 normal subjects. The stimulus, a whole-body roll of approximately 1 degrees, with the eye positioned 815 mm eccentric to the rotation axis, produced an inter-aural linear acceleration of approximately 0.5 g and a roll acceleration of approximately 360 degrees /s(2). The responses generated by the LVOR comprise horizontal eye rotations. Horizontal eye velocity at 100 ms from stimulus onset in UVD subjects was significantly lower than in normal subjects for all viewing distances, with no significant difference between ipsilesional and contralesional responses. LVOR acceleration gain, defined as the slope of actual horizontal eye velocity divided by the slope of ideal horizontal eye velocity during a 30-ms period starting 70 ms from stimulus onset, was bilaterally significantly reduced in UVD subjects at all viewing distances. Acceleration gain from all viewing distances was 1.04 +/- 0.28 in normal subjects, and in UVD subjects was 0.49 +/- 0.23 for ipsilesional and 0.63 +/- 0.27 for contralesional acceleration. LVOR enhancement in the first 100 ms by near viewing was still present in UVD subjects. LVOR latency in UVD subjects (approximately 39 ms) was not significantly different from normal subjects (approximately 36 ms). After UVD, LVOR is bilaterally and largely symmetrically reduced, but latency remains unchanged and modulation by viewing distance is still present.

Inferior vestibular neuritis.

Sudden, spontaneous, unilateral loss of vestibular function without simultaneous hearing loss or brain stem signs is generally attributed to a viral infection involving the vestibular nerve and is called acute vestibular neuritis. The clinical hallmarks of acute vestibular neuritis are vertigo, spontaneous nystagmus, and unilateral loss of lateral semicircular function as shown by impulsive and caloric testing. In some patients with vestibular neuritis the process appears to involve only anterior and lateral semicircular function, and these patients are considered to have selective superior vestibular neuritis. Here we report on two patients with acute vertigo, normal lateral semicircular canal function as shown by both impulsive and caloric testing, but selective loss of posterior semicircular canal function as shown by impulsive testing and of saccular function as shown by vestibular evoked myogenic potential testing. We suggest that these patients had selective inferior vestibular neuritis and that contrary to conventional teaching, in a patient with acute spontaneous vertigo, unilateral loss of lateral semicircular canal function is not essential for a diagnosis of acute vestibular neuritis.

Impulsive testing of individual semicircular canal function.

In order to test the human angular vestibulo-ocular reflex in the dynamic range of normal head movements, we measured 3-dimensional compensatory eye-movement responses to low-amplitude (10-12 degrees), high-acceleration (3000-4000 degrees/s/s), passive, manually delivered head rotations (head "impulses") in the three planes of the semicircular canals in normal subjects, in subjects who had recovered from surgical unilateral vestibular deafferentation, and in patients after acute unilateral peripheral vestibulopathy, that is, from vestibular "neuritis." We found that canal-plane head impulses away from an intact semicircular canal, that is, toward a lesioned semicircular canal, invariably produce a vestibulo-ocular reflex with permanently low gain, typically less that 0.4 if the lesion is complete. These results are a necessary consequence of primary semicircular canal afferents being driven into inhibitory saturation by rapid angular accelerations. With practice, clinicians can learn to recognize the telltale compensatory saccades that patients with unilateral loss of semicircular canal function will make if asked to look at an earth-fixed target during head impulses in any one of the three semicircular canal planes.

Enzyme kinetics determined using calorimetry: a general assay for enzyme activity?

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.

Catalytic efficiency and vitality of HIV-1 proteases from African viral subtypes.

The vast majority of HIV-1 infections in Africa are caused by the A and C viral subtypes rather than the B subtype prevalent in the United States and Western Europe. Genomic differences between subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. Because some amino acid polymorphisms occur at sites that have been associated with drug resistance in the B subtype, it is important to assess the effectiveness of protease inhibitors that have been developed against different subtypes. Here we report the enzymatic characterization of HIV-1 proteases with sequences found in drug-naive Ugandan adults. The A protease used in these studies differs in seven positions (I13V/E35D/M36I/R41K/R57K/H69K/L89M) in relation to the consensus B subtype protease. Another protease containing a subset of these amino acid polymorphisms (M36I/R41K/H69K/L89M), which are found in subtype C and other HIV subtypes, also was studied. Both proteases were found to have similar catalytic constants, k(cat), as the B subtype. The C subtype protease displayed lower K(m) values against two different substrates resulting in a higher (2.4-fold) catalytic efficiency than the B subtype protease. Indinavir, ritonavir, saquinavir, and nelfinavir inhibit the A and C subtype proteases with 2.5-7-fold and 2-4.5-fold weaker K(i)s than the B subtype. When all factors are taken into consideration it is found that the C subtype protease has the highest vitality (4-11 higher than the B subtype) whereas the A subtype protease exhibits values ranging between 1.5 and 5. These results point to a higher biochemical fitness of the A and C proteases in the presence of existing inhibitors.

Thermodynamic dissection of the binding energetics of KNI-272, a potent HIV-1 protease inhibitor.

KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.

Thermodynamic basis of resistance to HIV-1 protease inhibition: calorimetric analysis of the V82F/I84V active site resistant mutant.

One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. The active site mutant V82F/I84V has been shown to lower the binding affinity of protease inhibitors in clinical use. To identify the origin of this effect, we have investigated the binding thermodynamics of the protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir to the wild-type HIV-1 protease and to the V82F/I84V resistant mutant. The main driving force for the binding of all four inhibitors is a large positive entropy change originating from the burial of a significant hydrophobic surface upon binding. At 25 degrees C, the binding enthalpy is unfavorable for all inhibitors except ritonavir, for which it is slightly favorable (-2.3 kcal/mol). Since the inhibitors are preshaped to the geometry of the binding site, their conformational entropy loss upon binding is small, a property that contributes to their high binding affinity. The V82F/I84V active site mutation lowers the affinity of the inhibitors by making the binding enthalpy more positive and making the entropy change slightly less favorable. The effect on the enthalpy change is, however, the major one. The predominantly enthalpic effect of the V82F/I84V mutation is consistent with the idea that the introduction of the bulkier Phe side chain at position 82 and the Val side chain at position 84 distort the binding site and weaken van der Waals and other favorable interactions with inhibitors preshaped to the wild-type binding site. Another contribution of the V82F/I84V to binding affinity originates from an increase in the energy penalty associated with the conformational change of the protease upon binding. The V82F/I84V mutant is structurally more stable than the wild-type protease by about 1.4 kcal/mol. This effect, however, affects equally the binding affinity of substrate and inhibitors.

Fluoride inhibition of Klebsiella aerogenes urease: mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor.

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze the hydrolysis of urea. Here, we describe the steady-state and pre-steady-state kinetics of urease inhibition by fluoride. Urease is slowly inhibited by fluoride in both the presence and absence of substrate. Steady-state rate studies yield parallel double-reciprocal plots; however, we show that fluoride interaction with urease is not compatible with classical uncompetitive inhibition. Rather, we propose that fluoride binds to an enzyme state (E) that is in equilibrium with resting enzyme (E) and produced during catalysis. Fluoride binding rates are directly proportional to inhibitor concentration. Substrate reduces both the rate of fluoride binding to urease and the rate of fluoride dissociation from the complex, consistent with urea binding to E and E.F in addition to E. Fluoride inhibition is pH-dependent due to a protonation event linked to fluoride dissociation. Fluoride binding is pH-independent, suggesting that fluoride anion, not HF, is the actual inhibitor. We assess the kinetic results in terms of the known protein crystal structure and evaluate possible molecular interpretations for the structure of the E state, the site of fluoride binding, and the factors associated with fluoride release. Finally, we note that the apparent uncompetitive inhibition by fluoride as reported for several other metalloenzymes may need to be reinterpreted in terms of fluoride interaction with the corresponding E states.

HIV-1 protease inhibitors: enthalpic versus entropic optimization of the binding affinity.

Existing experimental as well as computational screening methods select potential ligands or drug candidates on the basis of binding affinity. Since the binding affinity is a function of the enthalpy (DeltaH) and entropy (DeltaS) changes, it is apparent that improved binding can be achieved in different ways: by optimizing DeltaH, DeltaS, or a combination of both. However, the behavior of enthalpically or entropically optimized inhibitors is fundamentally different, including their response to mutations that may elicit drug resistance. In the design of HIV-1 protease inhibitors, high binding affinity has usually been achieved by preshaping lead compounds to the geometry of the binding site and by incorporating a high degree of hydrophobicity. The thermodynamic consequence of that approach is that the binding affinity of the resulting inhibitors becomes entropically favorable but enthalpically unfavorable. Specifically, the resulting high binding affinity is due to an increased solvation entropy (hydrophobic effect) combined with a reduced loss of conformational entropy of the inhibitor upon binding (structural rigidity). Here we report that tripeptide inhibitors derived from the transframe region of Gag-Pol (Glu-Asp-Leu and Glu-Asp-Phe) bind to the HIV-1 protease with a favorable enthalpy change. This behavior is qualitatively different from that of known inhibitors and points to new strategies for inhibitor design. Since the binding affinities of enthalpically favorable and enthalpically unfavorable inhibitors have opposite temperature dependence, it is possible to design fast screening protocols that simultaneously select inhibitors on the basis of affinity and enthalpy.

Real-time rotation vectors.

Rotation vectors are a useful way of describing eye position without reference to arbitrary axes of rotation since any eye position can be reached from the reference position by rotation about a single axis. A real-time display of rotation vectors would not only help to acquire more reliable data, but would also widen the range of possible eye movement experiments. We describe a novel PC based data acquisition and analysis system which calculates and displays rotation vectors, velocity vectors and Listing's plane in real-time using voltages obtained from a two field coil system. The system was implemented using LabVIEW and optimised using Code Interface Nodes. Off-line processing can be sped up by varying parameters that indicate the amount of available RAM. During processing Listing's plane data can be rotated horizontally, vertically and torsionally. A computer controlled laser target changes position randomly every half second and so the targets are evenly spread, producing an appropriate range of eye positions which are used to calculate Listing's plane.

Head impulses reveal loss of individual semicircular canal function.

We studied individual semicircular canal responses in three dimensions to high-acceleration head rotations ("head impulses") in subjects with known surgical lesions of the semicircular canals, and compared their results to those of normal subjects. We found that vestibular-ocular reflex (VOR) gains at close to peak head velocity in response to yaw, pitch and roll impulses were reliable indicators of semicircular canal function. When compared to normals, lateral canal function showed a 70-80% decrease in VOR gain at peak of yaw head velocity during ipsilesional yaw impulses. After the loss of one vertical canal function there was a 30-50% decrease in vertical and torsional VOR gain in response to ipsilesional pitch and roll impulses respectively. Bilateral deficits in anterior or posterior canal function resulted in a 80-90% decrease in vertical VOR gain during ipsilesional pitch impulses, while the loss of ipsilateral anterior and posterior canal functions will result in a 80-90% decrease in torsional VOR gain in response to ipsilesional roll impulses. Three-dimensional vector analysis and animation of the VOR responses in a unilateral vestibular deafferented subject to yaw, pitch and roll impulses further demonstrated the deficits in magnitude and direction of the VOR responses following the loss of unilateral lateral, anterior and posterior canal functions.

The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease.

The effects of the peptide inhibitor acetyl pepstatin on the structural stability of the HIV-1 protease have been measured by high sensitivity calorimetric techniques. At 25 degrees C and pH 3.6, acetyl pepstatin binds to HIV-1 protease with an affinity of 1.6 x 10(7 )M-1 and an enthalpy of 7.3 +/- 0.5 kcal/mol, indicating that binding is not favored enthalpically and that the favorable Gibbs energy originates from a large positive entropy. Since the binding of acetyl pepstatin is associated with a negative change in heat capacity (-450 cal/K*mol) the association reaction becomes enthalpically favored at temperatures higher than 40 degrees C. The presence of the inhibitor stabilizes the dimeric structure of the protease in a fashion that can be quantitatively described by a set of thermodynamic linkage equations. The combination of titration and differential scanning calorimetry provides an accurate way of determining binding constants for high affinity inhibitors that cannot be determined by titration calorimetry alone. A structure-based thermodynamic analysis of the binding process indicates that the stabilization effect is not distributed uniformly throughout the protease molecule. The binding of the inhibitor selectively stabilizes those conformational states in which the binding site is formed, triggering a redistribution of the state probabilities in the ensemble of conformations populated under native conditions. As a result, the stability constants for individual residues do not exhibit the same change in magnitude upon inhibitor binding. Residues in certain areas of the protein are affected significantly whereas residues in other areas are not affected at all. In particular, inhibitor binding has a significant effect on those regions that define the binding site, especially the flap region which becomes structurally stable as a result of the additional binding free energy. The induced stabilization propagates to regions not in direct contact with the inhibitor, particularly to the strand between residues Pro9 and Ala22 and the helix between Arg87 and Gly94. On the other hand, the stability of the strand between Asp60 and Leu76 is not significantly affected by inhibitor binding. The structural distribution of binding effects define cooperative pathways within the protease molecule. Proteins 1999;36:147-156.