images of single, isolated proteins are
not a routine matter. This is because
an individual protein is often not astightly bound to a surface and lacks
the cooperative effects gained even by
the nonspeciﬁc binding existing in thelarger assemblies. Attempts to use
speciﬁc links to bind the molecule ofinterest to a ﬂat, solid substrate carry
the danger of forcing the protein into a
constrained geometry, which can mask
the structure-function relationships
being sought.
In this application note, a protocol
is demonstrated for characterizing
the ﬂexible structure of pro-MMP-9
using SPM. The results of thesemicroscopic studies were used as input
and ﬁne-tuning for SAXS models. The
combination of imaging and scattering
techniques, together with hydrodynamicstudies, yielded a consistent modelfor this enzyme showing clearly two
terminal domains joined by a ﬂexible
linker. In addition, SPM provided rather
unique information on possible protein
conformations adapted by the enzyme
upon its interaction with surfaces.
Comparison with a mutant lacking thislinker revealed the important role of thelinker domain.
SUBSTRATES FOR ENZYME BINDING

Smooth single-crystal silicon or mica
are ideal substrates on which to image
small molecules, due to the ease withwhich featureless, ultra-ﬂat surfaces
can be formed. In addition to thephysical characteristics, we requirethat the substrates bind the protein

protein
NHNH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

Gluteraldehyde
LinkerFigure 1. Schematic of aminized surface showing how the protein binds through the gluteraldehyde linker.

securely without altering or conﬁning
its natural shape. Amine-modiﬁcationof substrates has been shown to resultin an accessible covalent bonding
through the glutaraldehyde linkage,
without destroying the smoothnessof the underlying substrate.3, 4 In thiswork, we made use of amine-modiﬁed
silicon surfaces (see ﬁgure 1) as thesubstrate of choice. The plasma process
employed did not alter the smoothnessof the surface, and furthermore allowed
control of the surface
amine concentration.
Ideally, high-resolution images acquired
under buffer solution would provide theclosest approximation to the moleculein its natural state. Unfortunately, thefully hydrated molecule could not be
imaged sharply, as seen in ﬁgure 2A.
This phenomenon may arise because
the loose binding of the protein to thesurface possibly still allows some degreeof motion, or it could be related to thefact that the hydrated sample is softerand easier to distort under tip pressure.
Highly dehydrated samples, achievedby placing the sample in a vacuum
desiccator overnight, likewise resulted inpoor quality images (see ﬁgure 2B).
Best image quality was obtained
under, ambient conditions whereby thesample was rinsed and excess water
removed by 2 to 3 minutes of gentle
nitrogen ﬂow. Thus, sample volumesof 100 microliters, containing 0.1
milligrams per milliliter monodisperse
solution of pro-MMP9ΔOG mutant or
wild-type pro-MMP-9, fractionatedto contain the monomeric form, wereincubated on the amine-modiﬁed silicondies for 3 hours. The samples wererinsed gently with 2×200 microliters
buffer, followed by 5x200 microliters
Milli-Q water, and ﬁnally dried under
the stream of nitrogen. This procedure
likely leaves a thin hydration layer on thesample and is hence termed “semi-drymode.” The importance of this hydration
layer in atomic force microscopy (AFM)
imaging is suggested to result in images
representative of the fully
hydrated molecule.5, 6
In any event, when working with a driedsample, it is necessary that it be cleanof debris and dried salt deposited fromthe accompanying buffer solution. The
amine modiﬁcation provided sufﬁcientbinding of the protein so that buffer salts
could be readily rinsed away, without
removing the enzyme from the surface(see ﬁgure 2C). XPS measurements
veriﬁed the presence of the amine ata surface concentration of 4.7%. At
this level of amine concentration, andFigure 2. AFM images of pro-MMP-9. (A) Wild-type pro-MMP-9 under buffer solution, using oxide-sharpened silicon nitride tip. (B) Dessicated sample of the wildtype enzyme scanned in ambient conditions with spike tip. (C) Blank sample subjected to the same immobilization procedure but without applying the enzyme (spiketip). The arrow indicates the only particle observed on 1x1μm2 scan. Height scale of the color bar ranges from 0 to 50Å (dark to light). (Reprinted with permission
from reference 7.)
PAGE 2Using SPM to Visualize Enzymes at Submolecular Resolution

considering the molecular footprint,
there are statistically no more thanfour such bonds per protein molecule,which furthermore would not form
simultaneously as the molecule
approaches the substrate. From thiswe conclude that the binding itself
cannot signiﬁcantly constrain the protein
conformation. Horseradish peroxidase
assays showed that increasing theamine surface coverage beyond thislevel did not enhance binding.
AFM IMAGING AND ANALYSIS

Samples were imaged in air or inbuffer using TappingModeTM. In order tominimize the amount of force applied,
the amplitude set point was adjusted
to the maximum value that gave astable trace. High-resolution images of
biological samples in air were obtainedusing diamond-like carbon “spike” tips
with a rated tip radius of curvature of
2 nanometers or less. These probes
are only suitable for measurements onsurfaces with an RMS roughness of lessthen 20 nanometers due to the presenceof additional “spikes” on the probe that
could cause multiple contacts on arough surface. Oxide-sharpened silicon
nitride probes (Veeco DNP-S probes)
with nominal radius of 20 nanometers

were used for the liquid measurements,
which were performed in the standard
Veeco MultiMode® AFM liquid cell.
The sizes of the protein molecules
were determined from cross-sectional
analysis. The width values were thancorrected for broadening by the tip, bysubtracting the tip envelope as observed
from a typical high-resolution electron
microscope image.
RESULTS

The sample preparation proceduredescribed above assured that >95% ofthe features observed (approximately
20 in a 1×1 square micron image)represented the enzyme. Typical imagesof the wild-type and mutant enzyme
are shown in ﬁgure 3. The image crosssection indicates the existence of two
separated protein domains, which in themodel structure would be connected by
the OG linker (see ﬁgure 3C). In contrast,
the pro-MMP-9ΔOG mutant lacking the64-residue OG domain exhibits a rather
spherical shape with unresolved
domain separation.
The overall results are summarized in
the histograms of ﬁgure 4, which show
the height and width distributions of thetwo different samples. The two enzyme

species are easily distinguishable
in that the spread in both width andheight values for the wild type issigniﬁcantly larger than for the mutant.
Such differences could arise from theadditional degrees of freedom lent to thewild-type structure by the OG domain,
as opposed to the mutant, where thetwo lobes are more conﬁned. The
heterogeneity of sizes results from two
main factors, the different orientationsof the protein on the surface, anddifferent protein conformations. The
mutant has reduced conformational
degrees of freedom because it lacks theOG domain, so in this case the spreadof values stems mainly from different
orientations on the surface.
These AFM results are consistent
with the reported SAXS analysis, andreveal the details of the elongatedmulti-domain structure of pro-MMP-9.
The AFM results, together withhydrodynamic measurements, placed
additional constraints on the possible
molecular conformations consistent with
the SAXS measurements, and provided
a more concise view of the molecularstructure. Figure 5 shows a composite of
the AFM image with themolecular model.

Z-Axis Distance (Å)

2520151050050
100
X-Axis Distance (Å)

050
100
X-Axis Distance (Å)

150

Z-Axis Distance (Å)

2520151050150

Figure 3. AFM images of wild-type (A-C) and mutated (D-F) pro-MMP-9. All scans employed a spike tip. The cross-sections in (C) and (F) represent the dotted lines
in the 2D images in (A) and (D), respectively, and clearly show the difference in domain structure for the wild-type and mutant species. Height scale of the color bar
ranges from 0 to 50Å (dark to ligh). (Reprinted with permission from reference 7.)

Using SPM to Visualize Enzymes at Submolecular Resolution

PAGE 3